Mucosal cytotoxic T lymphocyte responses

ABSTRACT

The invention provides methods for induction of an antigen-specific, mucosal cytotoxic T lymphocyte response useful in preventing and treating infections with pathogens that gain entry via a mucosal surface.

RELATED APPLICATIONS

The present application is a continuation-in-part application of, andclaims the benefit under Title 35 of U.S. Provisional Application Nos.60/058,523 filed on Sep. 11, 1997, and No. 60/074,894 filed on Feb. 17,1998.

TECHNICAL FIELD

The present invention relates to methods and compositions forstimulating immune responses in mammals. More particularly, theinvention relates to methods and compositions for stimulating mucosalimmunity.

BACKGROUND OF THE INVENTION

Many infectious pathogens, e.g., HIV-1, enter their mammalian hosts viaa mucosal tissue prior to establishing a systemic infection. Veazey, etal., Science 280:427-431, 1998. Accordingly, vaccines capable ofprotecting against HIV should be capable of inducing long-term mucosalimmune responses. A number of recent studies have shown that such immuneresponses require direct stimulation of mucosal tissues, and may beachieved with live attenuated virus, Cranage, et al., Virology229:143-154, 1997, subunit SIV envelope Lehner, et al., Nature Medicine2:767-775, 1996, HIV-recombinant viruses, including recombinant MVA 89.6env (Belyakov et al., unpublished), or HIV peptide constructs Belyakov,et al., Proc. Nat. Acad. Sci. 95:1709-1714, 1998 (see also, Gallichan,et al., J. Exp. Med. 184:1879-1890, 1996; Cranage, et al., Virology229:143-154, 1997; and Rosenthal, et al., Semin. Immunol. 9:303-314,1997).

Numerous questions remain, however, concerning which vaccine candidatesmay afford the most effective protection against mucosal challenge withvirus, and what mechanisms may be involved in mediating protectiveimmunity. While a number of studies have shown a role for CTL inprotection against infections such as influenza that have a mucosalcomponent (Taylor and Askonas, Immunology 58:417-420, 1986; Epstein etal., J. Immunol. 160:322-327, 1998; Kulkarni et al., J. Virol.69:1261-1264, 1995), these reports have not established whether the CTLneed to be in a local mucosal site to protect. Conversely, while otherstudies have shown the induction of CTL in the mucosa, they have notestablished that these cells have a role in protection (Gallichan andRosenthal, J. Exp. Med. 184:1879-1890, 1996; Bennink et al., Immunology35:503-509, 1978; Lohman et al., J. Immunol. 155:5855-5860, 1995); andKlavinskis, et al., J. Immunol. 157:2521-2527, 1996 J. Immunol.155:5855-5860, 1995. Yet other studies have shown the induction byvaccines of protective immunity in the mucosa, but in the face ofmultiple immune responses, have not been able to sort out whichresponses are involved in protection (Lehner et al., Nature Medicine2:767-775, 1996; Putkonen et al., J. Virol. 71:4981-4984, 1997; Milleret al., J. Virol. 71:1911-1921, 1997; Quesada-Rolander et al., AIDS ResHwn Retroviruses 12:993-999, 1996; Bender et al., J. Virol.70:6418-6424, 1996; Wang et al., Vaccine 5:821-825, 1997).

Thus, although the role of CTL in protection against mucosal infectionshas been of interest for decades, especially in the case of influenzavirus, prior investigations have failed to identify fundamentalmechanisms linking immune responses to protection. In this regard,because mucosal infection by virus induces a local IgA response, it hasbeen too readily assumed that this response, and not a concomitant CTLresponse, was responsible for protection against viral infection throughthe mucosal route. However, the role of secretary IgA in neutralizingand protecting against mucosal HIV challenge is also not clear.

CTL are crucial mediators of immunity to intracellular microorganismssuch as viruses as well as certain bacteria and protozoan parasites. CTLspecifically recognize “non-self” antigenic peptides bound to majorhistocompatibility complex (MHC) class I molecules on the surface of“target cells” and then kill the target cells expressing the non-selfantigenic peptides. Non-self polypeptides from which the non-selfpeptides are derived can be a) proteins encoded by intracellularmicrobes, b) host-encoded proteins whose expression is induced by amicrobe, or c) mutant host encoded proteins expressed by, for example,tumor cells.

Thus, generation of CTL responses in the inductive and the effectormucosal immune system may be important to establishing effectiveprotective immunity to intracellular microbial pathogens that establishinfection via the mucosal barriers. In some cases, administration ofantigens via parenteral routes (subcutaneous, intramuscular, intravenousor intraperitoneal, for example) either fails to induce mucosal immunityor does so extremely inefficiently.

As noted above, previous reports of mucosal immune responses elicited bymucosal challenge with viruses have disclosed that the latter inducesantiviral antibody responses, and in some cases CTL responses, in theintraepithelial lymphoid populations. Chen et al., J. Virol.71:3431-3436, 1997; Sydora, et al., Cell Inununol. 167:161-169, 1996.However, it is not clear if either of such responses is relevant toprotection against viral infection in general, or HIV infection inparticular. Additional studies have suggested a role for CTL inprotection against infections that involve the mucosa, such as influenzaor respiratory syncytial virus, Taylor et al., Immunology 58:417-420,1986; Epstein et al., J. Immunol. 160:322-327, 1998; Kulkarni et al., J.Virol. 69:1261-1264, 1995. However, these studies have not addressed thequestion of whether CTL must be present at the mucosal site ofinfection, or if their principal activity occurs systemically.

Accordingly, a need exists in the art to better define the roles andmechanisms of CTL in mediating immunity and to develop new tools formediating immune protection against HIV and other pathogens,particularly by conferring immune protection at mucosal sites where suchpathogens initially proliferate.

SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions forinducing a protective mucosal CTL response in a subject. The methods ofthe invention involve administering either a soluble antigen itself, ora polynucleotide encoding the soluble antigen, to a mucosal surface. Thesoluble antigens can be full length, naturally occurring polypeptides orfragments (i.e., peptides) derived from them. Peptides to beadministered can be any length less than that of the naturally occurringpolypeptide. They can be, for example, five to one hundred amino acidresidues long, preferably twenty to seventy five amino acid residueslong, more preferably twenty five to sixty amino acid residues long andmost preferably thirty to fifty amino acid residues long.

The soluble antigen is administered with an adjuvant at the mucosal siteor without an adjuvant. Adjuvants can be, for example, cholera toxin(CT), mutant CT (MCT), E. coli heat labile enterotoxin (LT) or mutant LTD1 (MLT). IL-12 and/or IFNγ can be administered with the soluble antigeneither in the presence or absence of an adjuvant. Alternatively, the twocytokines (IL-12 and/or IFNγ) can be administered systemically andseparately from the soluble antigen which is administered mucosally,optionally with adjuvant. Mucosal routes of administration include IR,intranasal (IN), intragastric (IG), intravaginal (IVG) or intratracheal(IT).

Soluble antigens can be derived from pathogenic viruses (e.g., HIV-1,influenza virus or hepatitis A virus), bacteria (e.g, Listeriamonocytogenes), protozoans (e.g., Giardia lamblia). Alternatively, thesoluble antigen can be a tumor-associated antigen, e.g., prostatespecific antigen produced by prostate tumor cells or tyrosinase producedby melanoma cells. Peptide antigens can be cluster peptide vaccineconstructs (CL WvAC). For example, an HIV-1 CLUVAC can include one ormore of the following sequences: EQMHEDIISLWDQSLKPCVKRIQRGPGRAFVTIGK(SEQ ID NO:1), KQIINMWQEVGKAMYAPPISGQIRRIQRGPGRAFVTIGK (SEQ ID NO:2),RDNWRSELYKYKVVKIEPLGVAPTRIQRGPGRAFVTIGK (SEQ ID NO:3),AVAEGTDRVIEVWQGAYRAIRHIPRRIRQGLERRIQRGPGRAFVTIGK (SEQ ID NO:4),DRVIEVVQGAYRAIRHIPRRIRQGLERRIQRGPGRAFVTIGK (SEQ ID NO:5),DRVIEVVQGAYRAIRRIQRGPGRAFVTIGK (SEQ ID NO:6),AQGAYRAIRHIPRRIRRIQRGPGPRAFVTIGK (SEQ ID NO:7),EQMHEDIISLWDQSLKPCVKRIHIGPGRAFYTTKN (SEQ ID NO:8),KQIINMWQEVGKAMYAPPISGQIRRIHIGPGRAFYTTKN (SEQ ID NO:9),RDNWRSELYKYKVVKIEPLGVAPTRIHIGPGRAFYTTKN (SEQ ID NO:10),AVAEGTDRVIEVVQGAYRAIRHIPRRIRQGLERRIHIGPGRAFYTTKN (SEQ ID NO:11),DRVIEVVQGAYRAIRHIPRRIRQGLERRIHIGPGRAFYTTKN (SEQ ID NO:12),DRVIEVVQGAYRAIRRIHIGPGRAFYTTKN (SEQ ID NO:13) orAQGAYRAIRHIPRRIRRIHIGPGRAFYTTKN (SEQ ID NO:14).

Preferably, the CLUVAC includes the amino acid sequence of SEQ ID NO:2,SEQ ID NO:9 or SEQ ID NO:12. Antigenic peptides can be longer than thelength specified by the SEQ ID NOS.recited herein, i.e., the peptide canbe extended by adding one or more (e.g., 5, 10, 15, 20) amino acids atthe amino and/or carboxy termini of the peptide with any given SEQ IDNO.

The invention also encompasses methods for inducing a protective mucosalCTL response in a subject in which the soluble antigen is delivered IR.Preferably, the level of CTL activity induced by IR immunization is atleast 10% greater than that induced by other routes of mucosaladministration (e.g., IN). More preferably, mucosal CTL activity inducedby IR immunization is at least 2-fold, more preferably, at least 5-fold,and most preferably, at least 10-fold greater than that induced by otherroutes of mucosal immunization (e.g., IN or IG).

Subjects to which the methods of the invention are applied are mammals,e.g., humans, non-human primates, cats or mice.

Also provided within the invention are immunogenic compositions forinducing a protective mucosal CTL response in a subject which areadapted for intrarectal administration. The compositions comprise apurified soluble antigen formulated for intrarectal delivery to therectum, colon, sigmoid colon, or distal colon. They may be, formulatedas a rectal enema, foam, suppository, or topical gel and generallycomprise a base, carrier, or absorption-promoting agent adapted forintrarectal delivery.

In more detailed aspects, the immunogenic compositions of the inventionmay include a rectal emulsion or gel preparation, preferably wherein thesoluble antigen is admixed with a homogenous emulsion or gel carrier,eg., a polyoxyethylene gel. Alternatively, the soluble antigen may beadmixed with a rectally-compatible foam.

In other preferred aspects, the immunogenic compositions of theinvention are formulated in a suppository. The suppository is comprisedof a base or carrier specifically adapted for intrarectal delivery ofthe antigen. Preferred bases may be selected from a polyethyleneglycol,witepsol H15, witepsol W35, witepsol E85, propyleneglycol dicaprylate(Sefsol 228), Miglyol8lo, hydroxypropylcellulose-H (HPC), orcarbopol-934P (CP). More preferably, the suppository comprises at leasttwo base materials to optimize structural and delivery performance. Inother aspects, the suppository includes a stabilizing agent to minimizeintrarectal deradation of the soluble antigen.

To optimize intrarectal delivery, the immunogenic compositions of theinvention also preferably include an absorption-promoting agent, forexample a surfactant, mixed micelle, enamine, nitric oxide donor, sodiumsalicylate, glycerol ester of acetoacetic acid, cyclodextrin orbeta-cyclodextrin derivative, or medium-chain fatty acid.

In yet additional aspects of the invention, immunogenic compositions areprovided which include an adjuvant which enhances the CTL response.Suitable adjuvants are detoxified bacterial toxins, for exampledetoxified cholera toxin (CT), mutant cholera toxin (MCT), mutant- E.coli heat labile enterotoxin, and pertussis toxin. Preferably, theadjuvant is conjugated to a mucosal tissue or T cell binding agent, suchas protein A, an antibody that binds a mucosal tissue- orT-cell-specific protein, or a ligand or peptide that binds a mucosaltissue- or T-cell-specific protein. In more preferred aspects, theadjuvant is a recombinant cholera toxin (CT) having a B chain of CTsubstituted by protein A conjugated to a CT A chain, which exhibitsreduced toxicity and enhances mucosal tissue binding mediated by proteinA. Alternatively the adjuvant may be conjugated to a protein or peptidethat binds specifically to T cells, for example by binding CD4 or CD8(eg., the HIV V3 loop or a T cell-binding peptide fragment of the HIV V3loop).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar to those described herein can be used in the practice or testingof the present invention, suitable methods and materials are describedbelow. In case of conflict, the present application, includingdefinitions, will control. In addition, the materials, methods, andexamples described herein are illustrative only and not intended to belimiting other features and advantages of the invention, e.g.,prevention of viral or other infectious diseases, will be apparent fromthe following detailed description, from the drawings and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of line graphs showing the results of a CTL assay. IRHIV-1 peptide immunization induced long lasting mucosal and systemic CTLactivity.

FIG. 2 is a series of line graphs showing the results of a CTL assay. CTenhanced (but was not essential) for induction of CTL by IRadministration of antigenic peptide.

FIG. 3 is a series of line graphs showing the results of a CTL assay.CTL induced by IR immunization with an HIV-1 gp160 peptide specificallylysed target cells transfected with and expressing an HIV-1 gp160 gene.

FIG. 4 is a series of line graphs showing the results of a CTL assaywhich indicated that IR induction of CTL was IL-12 dependent.

FIG. 5 is a bar graph showing that IR immunization protects against IRchallenge with HIV-1 gp160 expressing recombinant vaccinia virus.

FIG. 6A is a line graph showing the results of a CTL assay using SPcells obtained from animals six months after IR immunization with anantigenic peptide.

FIG. 6B is a line graph showing the results of a CTL assay using SPcells obtained from animals six months after intranasal (IN)immunization with antigenic peptide.

FIG. 7A is a bar graph showing the results of a CTL F assay using PP aseffector cells.

FIG. 7B is a bar graph showing the results of a CTL assay using SP cellsas effector cells. PP and SP cells were obtained from animals thirtyfive days after mucosal (IR, IN, IG) or systemic (subcutaneous (SC))immunization with an antigenic peptide.

FIG. 8A is a bar graph showing the results of a CTL assay using effectorcells from wild-type BALB/c mice.

FIG. 8B is a bar graph showing the results of a CTL assay using effectorcells from BALB/c mice which lack the ability to produce functionalIFNγ. These experiments show the dependence of IR induction of CTL onIFNγ.

FIG. 9A is a line graph showing the results of a CTL assay using PP aseffector cells.

FIG. 9B is a line graph showing the results of a CTL assay using SPcells as effector cells. PP and SP cells were obtained from BALB/c micethirty five days after IR immunization with either antigenic peptide, CTand IL-12 (composition A) or antigenic peptide and CT (without IL-12;composition B).

FIGS. 10A and 10B demonstrate that IR immunization with PCLUS3-18IIIB orPCLUS3-18MN induces both Peyer's patch (panel A) and spleen (panel B)long-lasting immunity. However the levels of the induction of CTL aredifferent. Killing of P18IIIB-I10 (closed squares) or P18MN-T1Opeptide-pulsed targets (closed circles) is compared with killing ofunpulsed targets (open squares or circles). In both panel A and panel B,SEM of triplicate were all <5% of the mean.

FIG. 11 demonstrates that protection induced by mucosal immunizationwith HIV-1 peptide vaccine is specific. On day 35, mice were challengedintra-rectally with 2.5×10⁷ plaque-forming units (pfu) of vaccinia virusexpressing gp 160IIIB (vPE16) or with 2.5×10⁷ pfu of vaccinia virusexpressing β-galactosidase (vSC8). Bars=SEM of five mice per group. Thedifference is significant at P<0.01 by Student's test.

FIG. 12 demonstrates that protection induced by mucosal immunizationwith HIV-1 peptide vaccine is long-lasting. On day 35 or 6 months afterthe start of the immunization, mice were challenged intrarectally with2.5×10⁷ pfu of vaccinia virus expressing gp 160IIIB. Bars=SEM of fivemice per group. The difference is significant at P<0.01 by Student'stest.

FIG. 13 demonstrates that protection induced by mucosal immunizationwith HIV-peptide is dependent on CD8 positive T-cells. BALB/c mice weretreated IP with 0.5 mg monoclonal anti-CD8 antibody (clone 2.43, NIHFrederick, Md.) one day before and after each immunization and also twodays before and three days after the challenge with vPE16. Mice werechallenged intrarectally with 2×10⁷ pfu of vPE16 vaccinia virusexpressing gp 160IIIB.

FIGS. 14A and 14B demonstrate that mucosal immunization with HIV-1peptide induces mucosal CTL responses and stimulates protective immunityagainst intrarectal recombinant HIV-1 vaccinia challenge. FIG. 14Adepicts induction of the mucosal and systemic CTL responses by differentroutes of immunization with synthetic peptide HIV vaccine. Killing ofpeptide-pulsed targets (closed bars) is compared with killing ofunpulsed targets (open bars) at an effector-to-target ratio of 50:1.Panel A depicts results or immunizing on day 35 (IR or SC—bar 1, noimmunogen; bar 2, SC; bar 3, IR) BALB/c mice challenged intrarectallywith 2.5×10⁷ plaque-forming units (pfu) of vaccinia virus expressing gp160IIIB.

FIGS. 15A and 15B demonstrate enhancement of the mucosal (FIG. A) andsystemic (FIG. B) CTL responses to HIV-1 peptide by the mucosal (notsystemic) treatment with rmIL-12 BALB/C mice were treated by the IProute (right panels) or IR route (left panels) with 1 μg of the rmIL-12each day of the IR immunization with PCLUS3-18IIIB (50 μg/mice). On day35 HIV-specific Peyer's patch CTL (FIG. 15A) and spleen CTL (FIG. 15B)were studied.

FIG. 16 demonstrates that mucosal treatment with rmIL-12 in DOTAP alongwith HIV peptide vaccine enhances protection against mucosal challengewith vaccinia virus expressing gp160IIIB (vPE16). Five mice per groupwere immunized IR on days 0, 7, 14 and 21 with no immunogen (bar 1),with 50 μg PCLUS3-18IIB alone (bar 2), or with peptide plus 1 μg rmIL-12in DOTAP (bar 3), and challenged on day 35 intrarectally with 5×10⁷ pfuof vaccinia virus expressing gp 160IIIB. Viral pfu in the ovaries weredetermined six days later.

FIGS. 17A and 17B demonstrate that IL-12 cannot act directly in theinduction of mucosal CDB+CTL in the absence of IFNγ. IFNγ^(−/−) mice(BALB/c background) were immunized IR with the rmIL-12 (1μg/mouse)+DOTAP together with peptide. On day 35 HIV-specific Peyer'spatch CTL (FIG. 17A) and spleen CTL (FIG. 17B) were studied. Killing ofP18IIIB-I10 pulsed targets by effector cells from immunized IFNγ^(−/−)mice (closed squares) or conventional BALB/c mice (closed circles) iscompared with killing of unpulsed targets (open squares or circles).

DETAILED DESCRIPTION

A. Induction of Mucosal Immunity

The present invention is based on the discovery that IR administrationof a peptide, e.g., a synthetic multideterminant HXV-1 gp160 envelopeglycoprotein peptide, to a mucosal surface can induce anantigen-specific, protective CTL response in the mucosal immune system,even in the absence of a mucosal adjuvant.

The exemplary synthetic multideterminant peptides (CLUVAC) are composedof subregions containing epitopes that evoke some or all of (a) a helperT cell response, (b) a CTL response and (c) a high titer of neutralizingantibodies in multiple hosts of a given species expressing a broad rangeof MHC haplotypes. IR immunization with the HIV-1 CLUVAC PCLUS3-18IIIB(SEQ ID NO:2), and the mucosal adjuvant CT induced peptide-specific CTLin both the inductive (PP) and the effector (LP) sites of the mucosalimmune system, as well as in systemic lymphoid tissue, i.e., SP. Incontrast, systemic immunization with peptide vaccine produced HIV-1peptide-specific CTL only in the SP.

IR immunization induced long-lasting protective immune responses. Forexample, antigen-specific CTL were found in both mucosal and systemicsites 6 months after immunization. IR immunization with the antigenicpeptide elicited significantly stronger CTL responses than INimmunization with the same peptide. While IR administration withPCLUS3-18IIIB (SEQ ID NO:2) induced a significant response whenadministered alone, the response was enhanced by the inclusion of CT.The CTL were CD8⁺ T lymphocytes restricted by MHC class I molecules,recognizing MHC class I positive target cells either endogenouslyexpressing HIV-1 gp160 or′pulsed with an appropriate gp160 peptide.Induction of both mucosal and systemic CTL response by IR immunizationwas IL-12dependent, as shown by inhibition of induction of CTL in micetreated i.p. with anti-IL-12 antibody. Furthermore, inclusion of IL-12in the composition of antigenic peptide and CT used for IR immunizationresulted in enhanced mucosal and systemic CTL responses relative to theresponses elicited by antigenic peptide and CT without IL-12. Thedependence on IFNγ of mucosal and systemic CTL generation following IRinmnunization was demonstrated by the absence of such responses in micewhich lack the ability to produce functional IFNγ, e.g., as the resultof a premature stop-codon in the IFNγ-encoding gene. The stop-codonmutation causes the gene to encode a truncated protein lacking theactivity of IFNγ.

IR immunization with PCLUS3-18IIIB (SEQ ID NO:2) protected mice againstan IR challenge with a recombinant vaccinia virus expressing HIV-1 IIIBgp160. Thus, an HIV-1 peptide induced CTL responses in the mucosal andsystemic immune systems after IR immunization and protected againstmucosal challenge with virus expressing HIV-1 gp160.

The immunization method of the invention is useful to induce a mucosalCTL response to any soluble antigen. Accordingly, the invention providesa new method for vaccine administration to elicit immunologic protectionagainst viruses that enter through mucosal barriers, including HIV-1.The method can also be applied to achieving protection from infection bycertain bacteria (e.g., L. monocytogenes) and protozoa (e.g., G.lamblia) that establish infection via mucosae. In addition, sincemucosal immunization results in systemic generation of CTL activity, theprotocol can also be useful in prevention of infection by microorganismsthat enter through non-mucosal routes. The method is also useful forimmunotherapy of infections that are mucosally or non-mucosallyestablished. Finally, the methods can be utilized for immunotherapy ofcancer, both in the region of and remote from a mucosal surface.

B. Methods of Immunization

The invention features methods for protecting subjects from infection byintracellular microorganisms such as viruses as well as intracellularbacteria and protozoans. The methods involve induction of CTL responsesspecific for antigenic peptides derived from proteins encoded by genesof relevant microbes and expressed in association with MHC class Imolecules on the'surface of infected cells. This is achieved by deliveryof an appropriate soluble antigen to a mucosal surface, e.g., rectal,vaginal, nasopharyngeal, gastric or tracheal mucosae. Relevantmicroorganisms include but are not restricted to those that enter theirhosts via mucosal barriers, e.g. HIV-1, influenza virus, enteric virusessuch as rotaviruses, hepatitis A virus, papilloma virus, felineimmunodeficiency virus, feline leukemia virus, simian immunodeficiencyvirus, intracellular bacterial pathogens, e.g., L. monocytogenes andmycobacteria such as M. tuberculosis and M. leprae. Since the responseselicited by mucosal immunization occur in the systemic as well as themucosal immune system, the methods can also be applied to protectionfrom infection by intracellular pathogens that enter their hosts vianon-mucosal (e.g., HIV-1 in some scenarios such as a “stick” by acontaminated syringe needle, rabies virus and malarial protozoans) aswell as mucosal routes. In light of the above considerations,immunization via mucosae can also be used for immunotherapy ofintracellular infections.

Finally, the mucosal immunotherapy of the invention can be applied tosubjects with cancer, particularly, but not limited to, those with solidtumors in the region of the relevant mucosa, e.g., colonic, rectal,bladder, ovarian, uterine, vaginal, prostatic, nasopharyngeal, lung orcertain melanoma tumors. Tumor immunity is substantially mediated by CTLwith specificity for tumor associated peptides (e.g., prostate specificantigen peptides in prostatic cancer, carcinoembryonic antigen peptidesin colon cancer, human papilloma virus peptides in bladder cancer andMART1, gp100 and tyrosinase peptides in melanoma) (Rosenberg et al.(1994) J.N.C.I. 76:1159; Kawakami et al. (1994) Proc. Natl. Acad. Sci.USA. 91:3515) bound to MHC class I molecules on the surface of tumorcells.

B.1 Anticenic Polypeptides

A soluble antigen to be administered according to the invention can beany soluble carbohydrate or peptide antigen, e.g., one containing all orpart of the amino acid sequence of a peptide which is naturallyexpressed in association with a MHC class I molecule on the surface of acell infected with relevant microbe or expressed on a tumor cell. In thecase of infected cells, the cell surface expressed peptide is derivedfrom a protein either encoded by genes of the infectious agent or whoseexpression is induced by the infectious agent. Thus, the soluble antigencan be a full length, naturally occurring polypeptide, e.g., full lengthHIV-1 gp160 or gp120 or an antigenic fragment thereof.

Antigen-specific recognition by CTL involves interactions betweencomponents of the antigen-specific T cell receptor on the surface of theCTL and residues on both the antigenic peptide and the MHC class Imolecule to which the peptide is bound. Thus the soluble antigen canalso be a fragment (i.e., a peptide) of the naturally occurringpolypeptide that is either (a) itself capable of binding to MHC class Imolecules of multiple haplotypes on the surface of antigen presentingcells (APC) and stimulating CD8+ T cell responses in subjects expressingthese MHC class I haplotypes or (b) which can be proteolyticallyprocessed by APC into fragments with these properties. Ways ofestablishing the ability of a candidate peptide to stimulate a CTLresponse in the context of multiple MHC class I haplotypes are wellknown to one of ordinary skill in the art and are amply described inco-pending U.S. patent application Ser. No. 08/060,988 incorporatedherein by reference in its entirety.

Antigenic peptides can be engineered to bind to MHC class II moleculesof multiple haplotypes and will be recognized by CD4+ helper T cellprecursor cells of subjects expressing multiple MHC class II haplotypes.Ways of establishing the ability of a candidate peptide to stimulate ahelper T cell response in the context of multiple MHC class IIhaplotypes are well known to one of ordinary skill in the art and areamply described in co-pending U.S. patent application Ser. No.08/060,988 incorporated herein by reference in its entirety.

In addition, antigenic peptides, can contain epitopes that elicitneutralizing antibodies, i.e., those that bind to the relevant microbeor a cell infected with the microbe and neutralize or kill it. Ways ofestablishing these properties of a candidate peptide are well known toone of ordinary skill in the art and are amply described in copendingU.S. patent application Ser. No. 08/060,988 incorporated herein byreference in its entirety.

Antigenic peptides can also elicit antibodies that induceantibody-dependent cellular cytolysis (ADCC) of cells infected by theappropriate microbe or tumor cells expressing at their surface theprotein from which the antigenic peptide was derived. ADCC is aprotective mechanism by which specialized cells of the immune system (Kcells) recognize the Fc portion of IgG antibody molecules bound to thesurface of a target cell and lyse the relevant target cell. Sera, orother body fluids such as rectal lavages, from test subjects mucosallyimmunized with a candidate peptide can be tested for their ability tomediate ADCC by methods known to an ordinary artisan. A standard cellmediated lympholysis (CML) assay is used. Briefly, a source of lymphoidADCC effector cells (e.g., peripheral blood mononuclear cells (PBMC) orSP cells) is incubated in vitro with target cells expressing the abovedescribed cell surface protein in the presence of various dilutions oftest sera. Lysis of the target cells, which can be measured by therelease of a detectable label (⁵¹Cr, for is example) from prelabeledtarget cells, is an indication of the presence of ADCC inducingantibodies in the test serum.

Peptides of the invention can be cluster peptide vaccine constructs(CLUVAC). A CLLTVAC is a chimeric peptide containing a) a subregion withmultiple overlapping helper T cell activating epitopes that can bepresented by multiple MHC class II molecules (a cluster peptide), b) asubregion with a CTL activating epitope and c) a subregion that elicitsthe production of a neutralizing antibody. The peptide sequencescontaining these epitopes can be derived from different parts of amicrobial or tumor associated polypeptide. Alternatively, the CTLinducing and antibody neutralizing epitopes can be located in onesubregion of an antigen and the helper epitope(s) can be in a secondsubregion. CLUVAC and their design are extensively described inco-pending U.S. patent application Ser. No. 08/060,988 incorporatedherein by reference in its entirety.

HIV-1 CLUVAC can include the following sequences:EQMHEDIISLWDQSLKPCVKRIQRGPGRAFVTIGK (SEQ ID NO:1)KQIINMWQEVGKAMYAPPISGQIRRIQRGPGRAFVTIGK (SEQ ID NO:2)RDNWRSELYKYKYKIEPLGVAPTRIQRGPGRAFVTIGK (SEQ ID NO:3)AVAEGTDRVIEVWQGAYRAIRHIPRRIRQGLERRIQRGPGRAFVTIGK (SEQ ID NO:4)DRVIEVVQGAYRAIRHIPRRIRQGLERRIQRGPGRAFVTIGK (SEQ ID NO:5)DRVIEVVQGAYRAIRRIQRGPGRAFVTIGK (SEQ ID NO:6)AQGAYRAIRHIPRRIRRIQRGPGRAFVTIGK (SEQ ID NO:7)EQMHEDIISLWDQSLKPCVKRIHIGPGRAFYTTKN (SEQ ID NO:8)KQIINMWQEVGKAMYAPPISGQIRRIHIGPGRAFYTTKN (SEQ ID NO: 9)RDNWRSELYKYKVVKIEPLGVAPTRIHIGPGRAFYTTKN (SEQ ID NO:10)AVAEGTDRVIEVVQGAYRAIRHIPRRIRQGLERRIHIGPGRAFYTTKN (SEQ ID NO:11)DRVIEVVQGAYRAIRHIPRRIRQGLERRIHIGPGRAFYTTKN (SEQ ID NO:12)DRVIEVVQGAYRAIRRIHIGPGRAFYTTKN (SEQ ID NO:13)AQGAYRAIRHIPRRIRRIHIGPGRAFYTTKN (SEQ ID NO:14)

It is possible, for example, to link a particular cluster peptide to anypeptide containing a CTL and/or neutralizing antibody epitope. Examplesof cluster peptides include: cluster peptide 1 whose amino acid sequence(EQMHEDIISLWDQSLKPCVK) (SEQ ID NO:17) is the first 20 amino acids of theHIV-1 CLUVAC with SEQ ID NO:1; cluster peptide 3 whose amino acidsequence (KQIINMWQEVGKAMYAPPISGQIR) (SEQ, ID NO:18) is the first 24amino acids of the HIV-1 CLUVAC with SEQ ID NO: 2; cluster peptide 4whose amino acid sequence (RDNWRSELYKYKVVKIEPLGVAPT) (SEQ ID NO:19) isthe first 24 amino acids of the HIV-1 CLUVAC with SEQ ID NO:3; andcluster peptide 6 whose amino acid sequence(AVAEGTDRVIEWQGAYRAIRHIPRRIRQGLER) (SEQ ID NO:20) is the, first 33 aminoacids of the HIV-1 CLUVAC with SEQ ID NO:4.

Of particular interest are peptides containing the amino acid sequencesof SEQ ID NO:2, SEQ ID NO:9 and SEQ ID NO:12. Mucosal responses to thefirst (PCLUS3-18IIIB) are extensively characterized in the Examplespresented infra. PCLUS3-18MN (SEQ ID NO:9) and PCLUS6.1-18MN (SEQ IDNO:12) have been tested in human clinical trials. The CTE epitope withinPCLUS3-18IIIB (SEQ ID NO:2) is the amino acid sequence RIQRGPGRAFVTIGK(SEQ ID NO:15).

In some instances, mucosal adjuvants are co-administered at the mucosaltissue site with the soluble antigens. Such adjuvants include, but arenot limited to, detoxified bacterial toxins, for example detoxifiedcholera toxin (CT), E. coli heat labile toxin (LT), mutant CT (MCT)(Yamamoto et al. J. Exp. Med. 185:1203 (1997); Yamamoto et al. Proc.Natl. Acad. Sci. USA 58:5267 (1997); Douce et al. Infect. Immunity65:2821 (1997)), mutant E. coli heat labile toxin (MLT) (Di Tommaso etal. Infect. Immunity 64:974 (1996) Partidos et al. Immunology 89:83(1996), and pertussis toxin. MCT and MLT contain point mutations thatsubstantially ablate toxicity without substantially compromisingadjuvant activity relative to that of the parent molecules. In otherpreferred embodiments, the adjuvant is modified for increased binding tomucosal tissues and/or T-cells. Thus, in one aspect of the invention, CTor other bacterial toxins are conjugated to a mucosal tissue bindingagent, such as protein A, an antibody that binds a mucosal tissue- orT-cell-specific protein (eg., a receptor), or a ligand or peptide thatbinds a mucosal tissue- or T-cell-specific protein (eg., CD4 or CD8).For example, the B chain of CT may be substituted by protein A,conjugated to the CT A chain, to eliminate toxicity and enhance mucosaltissue binding mediated by protein A. Alternatively, the HIV V3 loopwhich binds CD4 on T cells may be conjugated to the adjuvant to enhancedelivery to T cells.

The CTL augmenting cytokines, eg., IL-12 and IFNγ, are, in someinstances, either administered systemically or, preferably,coadministered at the mucosal tissue site with the soluble antigen.

Variants of disclosed antigenic peptides can contain different aminoacids (preferably conservative changes) from the parental molecules butretain the biological activity of the parental molecules, e.g., theability to induce specific CTL responses subsequent to mucosalimmunization. Such variants can be synthesized by standard means, andare readily tested in assays known to those in the art. Antigenicpolypeptides are typically longer than the length of the nominal SEQ IDNOS. recited herein, i.e., the polypeptide can be extended by addingamino acids to the amino or carboxy termini of the peptide defined byany given SEQ ID NO.

Peptides and polypeptides of the invention will also include thosedescribed above but modified for in vivo use by:

(a) chemical or recombinant DNA methods to include mammalian signalpeptides (Lin et al., J. Biol. Chem. 270:14255 (1995)) or the bacterialpeptide, penetratin (Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864(1991)), that will serve to direct the peptide across cell andcytoplasmic membranes and/or traffic it to the endoplasmic reticulum(ER) of antigen presenting cells (APC), e.g., dendritic cells which arepotent CTL inducers;

(b) addition of a biotin residue which serves to direct the polypeptidesor peptide across cell membranes by virtue of its ability to bindspecifically to a translocator present on the surface of cells (Chen etal., Analytical Biochem. 227:168 (1995));

(c) addition at either or both the amino- and carboxy-terminal ends, ofa blocking agent in order to facilitate survival of the relevantpolypeptide or peptide in vivo. This can be useful in those situationsin which the termini tend to be degraded (“nibbled”) by proteases priorto cellular or ER uptake. Such blocking agents can include, withoutlimitation, additional related or unrelated peptide sequences that canbe attached to the amino and/or carboxy terminal residues of thepolypeptide or peptide to be administered. This can be done eitherchemically during the synthesis of the peptide or by recombinant DNAtechnology (see Section 3.3 infra). Alternatively, blocking agents suchas pyroglutamic acid or other molecules known to those of average skillin the art can be attached to the amino and/or carboxy terminalresidues, or the amino group at the amino terminus or carboxyl group atthe carboxy terminus replaced with a different moiety. Likewise, thepolypeptides or peptides can be covalently or noncovalently coupled topharmaceutically acceptable “carrier” proteins prior to administration.

Also of interest are peptidomimetic compounds based upon the amino acidsequence of the peptides of the invention. Peptidomimetic compounds aresynthetic compounds having a three-dimensional structure (i.e. a“peptide motif”) based upon the three-dimensional structure of aselected peptide. The peptide motif provides the peptidomimetic compoundwith the activity of binding to MHC molecules of multiple haplotypes andactivating CD8⁺ and CD4⁺ T cells from subjects expressing such MHCmolecules that is the same or greater than the activity of the peptidefrom which the peptidomimetic was derived. Peptidomimetic compounds canhave additional characteristics that enhance their therapeuticapplication such as increased cell permeability, greater affinity and/oravidity and prolonged biological half-life. The peptidomimetics of theinvention typically have a backbone that is partially or completelynon-peptide, but with side groups identical to the side groups of theamino acid residues that occur in the peptide on which thepeptidomimetic is based. Several types of chemical bonds, e.g. ester,thioester, thioamide, retroamide, reduced carbonyl, dimethylene andketomethylene bonds, are known in the art to be generally usefulsubstitutes for peptide bonds in the construction of protease-resistantpeptidomimetics.

B.2 In vivo Methods to Deliver Soluble Antigens to Mucosae of a Subject

In methods of the invention that induce mucosal CTL responses, a solubleantigen is delivered to antigen presenting cells of the inductivemucosal immune system (e.g., PP of the intestine). Delivery involvesadministering to a subject either the soluble antigen itself, e.g., anantigenic peptide, an expression vector encoding the soluble antigen, orcells transfected or transduced with the vector.

B.2.1 Administration of Soluble Antigen

Soluble antigens can be delivered to the mucosal immune system of amammal using techniques substantially the same as those described infrafor delivery to human subjects. Examples of appropriate mammals includebut are not restricted to humans, non-human primates, horses, cattle,sheep, dogs, cats, mice, rats, guinea pigs, hamsters, rabbits and goats.

A soluble antigen of the invention can be delivered to the mucosalimmune system of a human in its unmodified state, dissolved in anappropriate physiological solution, e.g., physiological saline.Alternatively, it can be modified as detailed in Section B.1 in order tofacilitate transport across cell and/or intracellular membranes and toprevent extracellular or intracellular degradation. Its transport acrossbiological membranes can also be enhanced by delivering it encapsulatedin liposomes using known methods (Gabizon et al., Cancer Res. 50:6371(1990); Ranade, J. Clin. Pharmacol. 29:685 (1989)) or an appropriatebiodegradable polymeric microparticle (also referred to as a“microsphere”, “nanosphere”, “nanoparticle, or “microcapsule”).Naturally, it is desirable that the soluble antigens be selectivelytargeted to the mucosae. This can be achieved by contacting the solubleantigens directly with the relevant mucosal surface, e.g., by IR, IVG,IN, intrapharyngeal (IPG) or IT infusion or implantation. CTL activity(systemic or mucosal) induced by IR immunization can be two-fold,preferably five-fold, more preferably twenty-fold, even more preferablyfifty-fold and most preferably two hundred-fold greater than thatinduced by IN immunization.

Soluble antigens of the invention can be delivered in liposomes intowhich have been incorporated ligands for receptors on relevant cells(e.g., dendritic cell or macrophage APC) or antibodies to cell-surfacemarkers expressed by these cells. Thus, for example, an antibodyspecific for a dendritic cell surface marker can direct liposomescontaining both the anti-dendritic cell antibody and the relevantsoluble antigen to dendritic cells.

The soluble antigens can be administered mucosally either alone ortogether with a mucosal adjuvant. Suitable adjuvants include, but arenot restricted to, CT, MCT and MLT. IL-12 and/or IFNγ can also beadministered to the subject. Thus, a soluble antigen can be administeredwith an adjuvant, with IL-12 (in the absence of an adjuvant), with IFNγ(in the absence of an adjuvant), with both IL-12 and IFNγ (in theabsence of an adjuvant), with an adjuvant and IL-12, with an adjuvantand IFNγ, or with an adjuvant and both IL-12 and IFNγ. The IL-12 andIFNγ can be administered systemically or co-administered mucosally withthe peptide. When the soluble antigen is administered encapsulated inliposomes or microparticles, the IL-12 and/or IFNγ can beco-incorporated into the same liposomes/microparticles or incorporatedinto separate liposomes/microparticles. Alternatively, the cytokines canbe administered in a free, soluble form. Examples of other cytokinesthat can be used, singly or in combination, are granulocyte-macrophagecolony-stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-7(IL-7), or tumor necrosis factor a (TNFa). These and otherimmunopotentiating cytokines are administered as discussed above forIL-12 and IFNγ. Administration can be single or multiple (two, three,four, five, six, eight or twelve administrations, for example). Wheremultiple, they can be spaced from one day to one year apart. When usingpeptides without a helper T-cell epitope,it can be necessary to carryout such multiple administrations.

In preferred aspects of the invention formulations of soluble antigenare prepared for intrarectal administration (i.e., delivery to therectum, colon, sigmoid colon, or distal colon), eg., by formulating thesoluble antigen in a rectal enema, foam, suppository, or topical gel.These rectal delivery formulations are adapted for improved delivery,stability, and/or absorption in the rectum, eg., by combining thesoluble antigen with one or more known intrarectal delivery base orcarrier materials, intrarectal absorption-promoting materials, and/orstabilizers.

Preferred base formulations for rectal delivery of soluble antigenwithin the methods of the invention include hydrophilic and hydrophobicvehicles or carriers such as those commonly used in formulatingsuppositories and rectal emulsion or gel preparations. Thus, emulsionvehicles are used which incorporate soluble antigen in an oil/wateremulsion suitable for intrarectal administration. Alternatively, gelformulations are provided which incorporate the soluble antigen in ahomogenous gel carrier, for example a polyoxyethylene gel such aspolyoxyethylene(20)cetylether(BC-20TX). When the antigen is formulatedin a rectally compatible foam, a non-CFC propellant foam is preferred.

Preferred carriers or delivery vehicles for use within the invention areconventional suppository base materials that are adapted for intrarectaluse, such as polyethyleneglycols. Exemplary polyethyleneglycols knownand available in art include PEG 400, PEG 1500, PEG 2000, PEG 4000 orPEG 6000. Preferred bases in this context include witepsol H15, witepsolW35, witepsol E85. These are selected for their desired lipophilicand/or hydrophilic properties, and are often selected to form a multiplelayer suppository with both lipophilic and hydrophilic base layers.Preferred lipophilic base materials are macregls of low molecularweight. Particular examples of suitable lipophilic bases includepropyleneglycol dicaprylate (Sefsol 228) and Miglyol810. A preferredhydrophilic base is PEG, eg., PEG 400. In one formulation useful withinthe invention, hydroxypropylcellulose-H (HPC) and/or carbopol-934P (CP)are used as bases for an inner suppository layer, and a witepsol base isused for the outer layer.

Crystalline cellulose or other common stabilizing agents can be added inselected amounts (eg., 30-60% by weight) to the base to promotesustained release.

Selection of base materials, stabilizers, and absorption-promotingagents for formulating intrarectal delivery compositions, particularlysuppositories, is determined according to conventional methods, eg.,based on melting and drop rates, breaking hardness, disintegration andspecial breaking times, spreading properties, and diffusion rates.Preferred values of these various properties are known and can bereadily determined to adjust antigen formulations to be suitable forintrarectal use. In particular, appropriate values for formulating atime-release suppository having appropriate structural and chemicalproperties for rectal delivery are known or readily ascertained. In thiscontext, conventional additives, eg., softeners such as neutral oils,Estasan, etc. may be included to optimize consistency, rate of deliveryand other characteristics of the formulation.

In formulating mucosal delivery compositions, it is also desired toinclude absorption-promoting agents to enhance delivery of the solubleantigen and, optionally the CTL-stimulatory cytokine, to the mucosalsurface. A variety of absorption promoting agents (i.e., agents whichenhance release or diffusion of the antigen and/or CTL-stimulatorycytokine from the delivery vehicle or base, or enhance delivery of theantigen and/or CTL-stimulatory cytokine to the mucosal tissue orT-cells, for example by enhancing membrane penetration) are known in theart and are useful in mucosal delivery formulations of the invention,eg., for inclusion in intrarectal delivery formulations, particularlysuppositories. These include, but are not limited to, surfactants (eg.,tween 80), mixed micelles, enamines, nitric oxide donors (eg.,S-nitroso-N-acetyl-DL-penicillamine, NOR1, NOR4—which are preferablyco-administered with an NO scavenger such as carboxy-PITO or doclofenacsodium), sodium salicylate, glycerol esters of acetoacetic acid (eg.,glyceryl-1,3-diacetoacetate or1,2-isopropylideneglycerine-3-acetoacetate) and otherrelease-diffusion/absorption-promoting agents adapted for mucosaldelivery.

Absorption-promoting agents useful within the invention include avariety of compounds specifically adapted for intrarectal use. In thiscontext, the rate and extent of rectal drug absorption are often lowerthan with oral absorption, possibly an inherent factor owing to therelatively small surface area available for drug uptake. In addition,the composition of rectal formulations (solid vs liquid, nature of thesuppository base) is an important factor in the absorption process. Thisrelationship between formulation and drug uptake has been clearlydemonstrated. Thus, coadministration of absorption-promoting agents(eg., surfactants, sodium salicylate, enamines) represents a keyapproach towards optimizing rectal drug absorption.

Rectal drug delivery in a site- and rate-controlled manner usingsuppositories, enemas, osmotic pumps, or hydrogel formulations providesa range of options for manipulating mucosal delivery, eg., controllingconcentrations, time-release, and immunogen-drug effects. Absorptionfrom aqueous and alcoholic solutions may occur very rapidly, butabsorption from suppositories is generally slower and very muchdependent on the nature of the suppository base, the use of surfactantsor other additives, particle size of the active ingredients, etc.

Accordingly, preferred formulations for administering soluble antigensand CTL-stimulatory cytokines within the methods of the invention aredesigned to optimize mucosal delivery. These agents may thus includecyclodextrins and beta-cyclodextrin derivatives (e.g.,2-hydroxypropyl-beta-cyclodextrin andheptakis(2,6-di-O-methyl-beta-cyclodextrin). These compounds, preferablyconjugated with one or more of the active ingredients and formulated inan oleaginous base, are well documented to enhance bioavailability inintrarectal formulations. Other absorption-enhancing agents adapted forintrarectal delivery include medium-chain fatty acids, including mono-and diglycerides (e.g., sodium caprate-extracts of coconut oil, Capmul),and triglycerides (e.g., amylodextrin, Estaram 299, Miglyol 810).

It is well known in the medical arts that dosages for any one humansubject depend on many factors, as well as the particular compound to beadministered, the time and route of administration and other drugs beingadministered concurrently. Dosages for the soluble antigens of theinvention will vary, but can be approximately 0.01 mg to 100 mg peradministration. Dosages for the mucosal adjuvants will be approximately0.001 mg to 100 mg per administration. Dosages for IL-12 will beapproximately 25 μg/kg to 500 μg/kg and for IFNγ will be 300 KU to30,000 KU per administration—comparable dosages will be used for othercytokines. For example, 3,000 KU of IFNγ can be administered to a humanpatient once per week. Methods of determining optimal doses are wellknown to pharmacologists and physicians of ordinary skill. Routes willbe, as recited supra, mucosal, e.g., IR, IG, IVG, IN, IPG or IT.

B.2.2 Administration of Soluble Antigens Utilizing Expression VectorsEncoding the Soluble Antigens.

An expression vector is composed of or contains a nucleic acid in whicha polynucleotide sequence encoding a peptide or polypeptide of theinvention is operatively linked to a promoter or enhancer-promotercombination. A promoter is a trancriptional regulatory element composedof a region of a DNA molecule typically within 100 nucleotide pairsupstream of the point at which transcription starts. Anothertranscriptional regulatory element is an enhancer. An enhancer providesspecificity in terms of time, location and expression level. Unlike apromoter, an enhancer can function when located at variable distancesfrom the transcription site, provided a promoter is present. An enhancercan also be located downstream of the transcription initiation site. Acoding sequence of an expression vector is operatively linked to atranscription terminating region. To bring a coding sequence undercontrol of a promoter, it is necessary to position the translationinitiation site of the translational reading frame of the peptide orpolypeptide between one and about fifty nucleotides downstream (3′) ofthe promoter. Examples of particular promoters are provided infra.Expression vectors and methods for their construction are known to thosefamiliar with the art.

Suitable vectors include plasmids, and viral vectors such as herpesviruses, retroviruses, vaccinia viruses, attenuated vaccinia viruses,canary pox viruses, adenoviruses and adeno-associated viruses, amongothers.

The application of antigen-encoding genes to the mucosal induction ofCTL in humans can utilize either in vivo or ex vivo based approaches.

The ex vivo method includes the steps of harvesting cells (e.g.,dendritic cells) from a subject, culturing the cells, transducing themwith an expression vector, and maintaining the cells under conditionssuitable for expression of the soluble antigen. These methods are wellknown in the art of molecular biology. The transduction step isaccomplished by any standard means used for ex vivo gene therapy,including calcium phosphate, lipofection, electroporation, viralinfection, and biolistic gene transfer. Cells that have beensuccessfully transduced are then selected, for example, for expressionof a drug resistance gene. The cells can then be lethally irradiated (ifdesired) and injected or implanted into the subject. IL-12 and IFNγ canbe administered either systemically or to the relevant mucosal surfaceas discussed above (Section B.2.1).

The in vivo approach requires delivery of a genetic construct directlyinto the mucosa of a subject, preferably targeting it to the cells ortissue of interest (e.g., dendritic cells in PP). This can be achievedby administering it directly to the relevant mucosa (e.g., IR in thecase of PP). Tissue specific targeting can also be achieved by the useof a molecular conjugate composed of a plasmid or other vector attachedto poly-L-lysine by electrostatic or covalent forces. Poly-L-lysinebinds to a ligand that can bind to a receptor on target cells (Cristianoet al. J. Mol. Med 73:479 (1995)). Similarly, cell specific antibodiesof the type described supra in Section B.2.1 can be bound to vectors andthereby target them to the relevant cells of the mucosal immune system.A promoter inducing relatively tissue or cell-specific expression can beused to achieve a further level of targeting. Appropriatetissue-specific promoters include, for example, the inducible IL-2(Thompson et al., Mol. Cell. Diol. 12: 1043 (1992)), IL-4 (Todd et al.,J. Exp. Med. 177:1663 (1993)) and IFNγ (Penix et al., J. Exp. Med.178:483 (1993)) T-cell targeting promoters. These promoters would allowproduction of the soluble antigens in lymphoid tissue, including mucosallymphoid tissue, e.g., PP. Naturally, an ideal promoter would be adendritic cell specific promoter.

Vectors can also be delivered by incorporation into liposomes or otherdelivery vehicles either alone or co-incorporated with cell specificantibodies, as described supra in Section B.2.1.

DNA or transfected cells can be administered in a pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers arebiologically compatible vehicles which are suitable for administrationto a human, e.g., physiological saline. A therapeutically effectiveamount is an amount of the DNA of the invention which is capable ofproducing a medically desirable result in a treated animal. As is wellknown in the medical arts, the dosage for any one patient depends uponmany factors, including the patient's size, body surface area, age, theparticular compound to be administered, sex, time and route ofadministration, general health, and other drugs being administeredconcurrently. Dosages will vary, but a preferred dosage foradministration of DNA is from approximately 10⁶ to 10¹² copies of theDNA molecule. This dose can be repeatedly administered, as needed.Routes of administration will be the mucosal routes recited for solubleantigens supra in Section B.2.1. The mucosal adjuvants, IL-12 and IFNγcan also be administered in the same combinations, by the same routesand at the same dosages recited in Section B.2.1.

B.3 Sources of Peptides and Polypeptide

Peptides and polypeptides used in the methods of the invention can beobtained by a variety of means. Smaller peptides (less than 100 aminoacids long) can be conveniently synthesized by standard chemical methodsfamiliar to those skilled in the art (e.g, see Creighton, Proteins:Structures and Molecular Principles, W.H. Freeman and Co., N.Y. (1983)).Larger peptides (longer than 100 amino acids) can be produced by anumber of methods including recombinant DNA technology (see infra). Somepolypeptides (e.g., HIV-1, gp160, CT, LT, IL-12, or IFNγ) can bepurchased from commercial sources.

Polypeptides such as HIV-1, gp160, CT, IL-12, or IFNγ, can be purifiedfrom biological sources by methods well-known to those skilled in theart (Protein Purification, Principles and Practice, second edition(1987) Scopes, Springer Verlag, N.Y.). They can also be produced intheir naturally occurring, truncated, chimeric (as in the CLUVAC, forexample), or fusion protein forms by recombinant DNA technology usingtechniques well known in the art. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. See, for example, the techniques described inSambrook et al. (1989) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Press, N.Y.,; and Ausubel et al., eds. (1989), CurrentProtocols in Molecular Biology, Green Publishing Associates, Inc., andJohn Wiley & Sons, Inc., N.Y. Alternatively, RNA encoding the proteinscan be chemically synthesized. See, for example, the techniquesdescribed in Oligonucleotide Synthesis, (1984) Gait, M. J. ed., IRLPress, Oxford, which is incorporated by reference herein in itsentirety.

A variety of host-expression vector systems can be utilized to expressthe nucleotide sequences. Where the peptide or polypeptide is soluble,it can be recovered from: (a) the culture, i.e., from the host cell incases where the peptide or polypeptide is not secreted; or (b) from theculture medium in cases where the peptide or polypeptide is secreted bythe cells, The expression systems also encompass engineered host cellsthat express the polypeptide in situ, i.e., anchored in the cellmembrane. Purification or enrichment of the polypeptide from such anexpression system can be accomplished using appropriate detergents andlipid micelles and methods well known to those skilled in the art.Alternatively, such engineered host cells themselves can be used insituations where it is important not only to retain the structural andfunctional characteristics of the protein, but also to assess biologicalactivity.

The expression systems that can be used for purposes of the inventioninclude but are not limited to microorganisms such as bacteria (forexample, E. coli and B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining the nucleotide sequences; yeast transformed with recombinantyeast expression vectors; insect cells infected with recombinant viralexpression vectors (baculovirus); plant cell systems infected withrecombinant viral expression vectors (e.g., cauliflower mosaic virus,CAMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors; or mammalian cells (e.g., COS, CHO, BHK, 293, 3T3)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses.

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the geneproduct being expressed. For example, when a large quantity of such aprotein is to be produced, e.g., for in vivo immunization, vectors whichdirect the expression of high levels of fusion protein products that arereadily purified can be desirable. Such vectors include, but are notlimited to, the E. coli expression vector pUR278, (Ruther et al., EMBOJ. 2:1791 (1983)), in which the coding sequence can be ligatedindividually into the vector in frame with the lacZ coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 1:3101 (1985); Van Heeke & Schuster, J. Biol. Chem. 264:5503(1989)); and the like. PGEX vectors can also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. The PGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene product can be released from the GST moiety.

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the nucleotide sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the gene product in infected hosts (e.g., See Logan andShenk, Proc. Natl. Acad. Sci. USA 81:3655 (1984)). Specific initiationsignals can also be required for efficient translation of insertednucleotide sequences. These signals include the ATG initiation codon andadjacent sequences. In cases where an entire gene or cDNA, including itsown initiation codon and adjacent sequences, is inserted into theappropriate expression vector, no additional translational controlsignals may be needed. However, in cases where only a portion of thecoding sequence is inserted, exogenous translational control signals,including, perhaps, the ATG initiation codon, must be provided.Furthermore, the initiation codon must be in phase with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression can be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc.(Bittner et al., Methods in Enzymol. 153:516 (1987)).

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Appropriate cell lines orhost systems can be chosen to ensure the correct modification andprocessing of the foreign protein expressed. Mammalian host cellsinclude but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293,3T3, and WI38.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe sequences described above can be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells can beallowed to grow for 1-2 days in an enriched medium, and then areswitched to a selective medium. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into their chromosomes and grow to form foci whichin turn can be cloned and expanded into cell lines. This method canadvantageously be used to engineer cell lines which express the geneproduct. Such engineered cell lines can be particularly useful inscreening and evaluation of compounds that affect the endogenousactivity of the gene product.

A fusion protein can be readily purified by autilizing an antibody or aligand that specifically binds to the fusion protein being expressed.For example, a system described by Janknecht et al., Proc. Natl. Acad.Sci. USA 88:8972 (1991) allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines. In thissystem, the gene of interest is subcloned into a vaccinia recombinationplasmid such that the gene's open reading frame is translationally fusedto an amino-terminal tag consisting of six histidine residues. Extractsfrom cells infected with recombinant vaccinia virus are loaded onto Ni₂₊nitriloacetic acid-agarose columns and histidine-tagged proteins areselectively eluted with imidazole-containing buffers. If desired, thehistidine tag can be selectively cleaved with an appropriate enzyme.

The following examples are meant to illustrate the invention and not tolimit it.

EXAMPLES

Materials and Methods

Human IR immunization Soluble antigens of the invention are dissolved ina pharmaceutically acceptable carrier, e.g., physiological saline andadministered with and without adjuvant and with or without cytokines,e.g., IL-12 and/or IFNγ. The latter can be administered systemically ortogether with the antigen. Dosages of soluble antigen are approximately0.001 mg to 100 mg per administration. Administration can be single, ormultiple. In the case of multiple immunizations, there can, for example,be four weekly administrations, followed (if desired) by a boosteradministration several months (e.g., two, three, four, six, eight ortwelve) or several years (e.g., two, three, four, five, ten, twenty orthirty) thereafter. Administration of the antigen (with and withoutadjuvant and with and without cytokine) can be by IR insertion of asuppository into which the immunizing composition has been incorporated,by enema or by flexible sigmoidoscope.

CTL activation cultures At various time points after immunization(different for each experiment), CTL activity was assessed. BALB/c micewere sacrificed and the intestines and SP were surgically removed.Single cell suspensions were prepared from the organs by methodsfamiliar to those of average skill in the art. In the case of PP and LP,the PP were first dissected from the outer surface of the intestine. Therest of the intestine was cut into small fragments 1 to 3 mm squarewhich were suspended in phosphate buffered saline (PBS). The tissuefragment suspension was stirred for 20 minutes at room temperature andshaken under the same conditions to liberate the intraepitheliallymphocytes (IEL) from the tissue. The suspended IEL were discarded andtissue fragments were treated with collagenase type VIII (Sigma) (300U/ml) dissolved in PBS for 1 hour at room temperature to release the LPcells. PP cells were extracted from the dissected PP by the samecollagenase treatment. Cells (PP or LP) were then placed on adiscontinuous gradient containing 75% and 40% of Percoll, followed bycentrifugation at 2,000 r.p.m. for 20 minutes. Lymphocytes wereharvested from the 75%/40% interface and washed two times. Immune cellsfrom SP, PP, LP were cultured with 1 μM P18IIIB-I10 peptide at 5×10⁶per/milliliter in 24-well culture plates in complete T cell medium(CTM): RPMI 1640 containing 10% fetal bovine serum, 2 mM L-glutamine,penicillin (100 U/ml), streptomycin (100 μg/ml), and 5×10⁻⁵ M2-mercaptoethanol. P18IIIB-I10 is a peptide containing the minimalessential CTL epitope of P18IIIB (SEQ ID NO:15) and has the sequence:RGPGRAFVTI (SEQ ID NO:16). Three days later, 10% supernatant fromconcanavalin A activated spleen cells was added as a source ofinterleukin-2 (IL-2). After 7 days of culture, LP lymphocytes (LPL) werestimulated in a second 7 day culture with 1 μM P18IIIB-I10 peptide (SEQID NO:16) together with 4×10⁶ of 3300-rad irradiated syngeneic SP cells.Immune SP and PP cells were similarly stimulated in vitro for two 7-dayculture periods before assay. Cytolytic activity of CTL was measured bya 4-hour assay with ⁵¹Cr labeled P815 targets using a method familiar tothose of ordinary skill in the art. For testing the peptide specificityof CTL, ⁵¹Cr-labeled P815 targets were pulsed for 2 hours with peptideat the beginning of the assay. The percent specific ⁵¹Cr release wascalculated as 100× (experimental release-spontaneous release)/(maximumrelease-spontaneous release). Maximum release was determined fromsupernatants of cells that were lysed by addition of 5% Triton-X 100. Asa control, spontaneous release was determined from target cellsincubated without added effector cells. Standard errors of the mean oftriplicate cultures were all <5% of the mean.

Viral Plague assay Six days after IR challenge with recombinant vacciniavirus expressing HIV-1 IIIB gp160 (vPE16), mice were sacrificed. Theirovaries were removed, dissociated into single cell suspensions andassayed for vPE16 titer by plating serial 10-fold dilutions on a plateof BSC-2 indicator cells, staining with crystal violet and countingplaques at each dilution. The minimal detectable level of virus was 70plaque forming units (pfu).

Example 1 Comparison of Mucosal and Systemic CTL Responses After Mucosaland Systemic Immunization

Mice were immunized IR with 4 doses (on days 0, 7, 14 and 21) of theHIV-1 CLUVAC PCLUS3-18IIIB (SEQ ID NO:2) (50 μg/mouse). 5 weeks to 6months after the first dose, antigen-specific T cells were isolated fromPP, LP and SP and tested for the presence of HIV-1 P18IIIB peptidespecific CTL (FIG. 1), as described above. Closed squares show killingof P18IIIB-I10 (SEQ ID NO:16)-pulsed targets and open diamonds showkilling on unpulsed targets. IR immunization induced long-lastingprotective immune responses: antigen-specific CTL were detected inmucosal inductive (PP) and effector (LP) sites and in a systemic site(SP) at least 6 months after immunization. In contrast, systemicimmunization (s.c. in incomplete Freund's adjuvant) induced CTL in thespleen but not in the mucosal immune system (i.e., PP and LP).

Example 2 Comparison of CTL Responses with and without a MucosalAdjuvant

BALB/c mice were immunized IR with 4 doses of the synthetic HIV-1 CLUVACPCLUS3-18IIIB (SEQ ID NO:2) (50 μg/mouse per immunization) alone, i.e.,in the absence of adjuvant or cytokine on days 0, 7, 14 and 21. Inparallel, another group of mice was immunized IR with PCLUS3-P18IIIB(SEQ ID NO:2) HIV-1 peptide in combination with CT (leg/mouse). On day35 antigen-specific T cells were isolated from PP, LP and SP. Immunecells from SP, PP, or LP were cultured and tested for antigen specificCTL (FIG. 2), as described above. Closed squares show killing ofP18IIIB-I10 (SEQ ID NO:16) pulsed targets, and open diamonds showkilling of unpulsed targets. IR administration of peptide alone induceda significant response. The response was enhanced by theco-administration of CT.

Example 3 CTL Induced by Mucosal Immunization Lyse Targets ExpressingHRV-1 gp160 Envelope Protein

Mice were immunized IR with 4 doses (on days 0, 7, 14 and 21) of theHIV-1 CLUVAC PCLUS3-18IIIB (SEQ ID NO:2) (5 μg/mouse per immunization)in the presence of CT (1 μg/mouse), on day 35, antigen-specific T cellswere isolated from PP, LP and SP. Immune cells from SP, PP, or LP werecultured as described above. Cytolytic activity of CTL was measuredusing a standard ⁵¹Cr release assay (FIG. 3).

Three different cell lines were used as target cells: 15-12 cells, 3T318 Neo BALB/c cells and P815 cells. 15-12 cells are BALB/c 3T3fibroblasts transfected with a vector encoding HIV-1 gp160 (Takahashi etal. Proc. Natl. Acad. Sci. USA 8:3105 (1988)); 3T3 18 Neo BALB/c cellsare BALB/c 3T3 fibroblasts transfected with an expression vectorcontaining a Neor gene but no gp160 gene; and P815 cells areuntransfected cells that present antigenic peptides added to theculture. CTL lysis of gp160 expressing 15-12 cells (closed squares inright panels) was compared to that of control gp160 non-expressing 3T318 Neo BALB/c cells (open diamonds right panels). P815 target cells(left panel) were tested in the presence (closed squares) or absence(open diamonds) of P18IIIB-I10 peptide (SEQ ID NO:16) (1 μM). Thepercent specific 51Cr release was calculated as described above. CTLinduced by IR immunization killed target cells either endogenouslyexpressing HIV-gp160 or pulsed with P18IIIB peptide (SEQ ID NO:15).

Example 4 CTL Induced by IR Immunization are IL-12 Dependent

BALB/c mice were immunized IR with 4 doses (on days 0, 7, 14 and 21) ofCLUVAC PCLUS3-18IIIB (SEQ ID NO:2) (50 μg/mouse per immunization) incombination with CT (1 μg/mouse). One day before and one day afterimmunization with peptide mice were treated intraperitoneally (i.p.)with anti-IL-12 antibody (0.5 mg/per injection: 4 mg/mouse total dose(FIG. 4, right panels) or were untreated (FIG. 4, left panels). On day35, antigen-specific T cells were isolated from PP, LP and SP. Immunecells from SP, PP, or LP were cultured as described above. Cytolyticactivity of CTL was measured (FIG. 4) as described above. For testingthe peptide specificity of CTL, S51Cr labeled P815 targets were pulsedwith peptide P18IIIB-I10 (SEQ ID NO:16) at the beginning of the assay(closed squares), or (as a control) left unpulsed i.e., without peptide(open diamonds). Induction of both mucosal and systemic CTL responses byIR immunization was IL-12-dependent, as shown by inhibition of inductionof CTL in mice treated i.p. with anti-IL-12 antibody.

BALB/c mice were immunized IR on days 0, 7, 14 and 21 with eithercomposition A containing the synthetic HIV-1 CLUVAC PCLUS3-18IIIB (SEQID NO:2) (50 μg/mouse per immunization), CT (10 μg/mouse perimmunization), and recombinant IL-12 (1 μg/mouse per immunization), orcomposition B containing the synthetic HIV-1 CLUVAC PCLUS3-18IIIB (SEQID NO:2) (50 μg/mouse per immunization), and CT (10 μg/mouse perimmunization), on day 35, antigen-specific T cells were isolated from PPand SP. Immune cells from PP (FIG. 9A) and SP (FIG. 9E) were separatelycultured and tested for antigen specific CTL, as described above. FIG.9A shows killing of peptide P18IIIB-I10 (SEQ ID NO:16) pulsed targetcells by effector cells from mice immunized with composition A (i.e.,antigen with IL-12) (open squares), and FIG. 9B shows killing is of theP18IIIB-I10 (SEQ ID NO:16) pulsed target cells by effector cells frommice immunized with composition B (i.e., antigen without IL-12) (opendiamonds). In both FIGS. 9A and 9B, control data was obtained using thetwo effector populations tested on unpulsed targets. The enhanced CTLresponses of both PP (FIG. 9A) and SP (FIG. 9B) effector cells from miceimmunized IR with composition A compared to the responses elicited by IRimmunization with composition B indicate that co-administration of IL-12augments mucosal and systemic CTL responses. These data both provideindependent evidence for the role of IL-12 in eliciting mucosal (andsystemic) immunity by mucosal administration of antigens and confirm thefindings of the antibody inhibition data described above.

Example 5 Intrarectal Peptide Immunization Protects Against MucosalChallenge with EIV-gp160 Expressing Recombinant Vaccinia Virus

Groups of 5 mice were immunized IR with 4 doses of the HIV-1 CLUVACPCLUS3-18IIIB (SEQ ID NO:2) (50 μg/mouse per immunization) on days 0, 7,14 and 21 in combination with CT. On day 35, mice were challenged IRwith 2.5×10⁷ pfu of vaccinia virus expressing gp160 IIIB (vPE16). After6 days, the mice were killed and their ovaries assayed for the presenceof virus (FIG. 5) as described above. The left bar of FIG. 5 shows virustiter in the ovaries of unimmunized mice and the right bar shows virustiter in the ovaries of immunized mice. IR immunization withPLCUS3-18IIIB (SEQ ID NO:2) protected mice against IR challenge withvPE16 as shown by a 4.5-log reduction in viral pfu in ovaries comparedto unimmunized animals (p<0.005).

Example 6 Comparison of Systemic CTL Responses Six Months After IR andIN Immunization

Mice were immunized IR or IN with 4 doses (on days 0, 7, 14 and 21) ofthe HIV-1 CLUVAC PCLUS3-18IIIB (SEQ ID NO:2) (50 μg/mouse). Six monthsafter the first dose, antigen-specific T cells were isolated from SP andtested for the presence of HIV-1 P18IIIB-I10 (SEQ ID NO:16) peptidespecific CTL (FIGS. 6A and 6B) as described above. Closed squares showkilling of P18IIIB-I10 (SEQ ID NO:16)-pulsed targets and open diamondsshow killing of unpulsed targets. The level of CTL activity induced byIR immunization (FIG. 6A) was higher than that induced by INimmunization (FIG. 6B). These data indicate that IR immunization inducedpotent, long-lasting, antigen-specific splenic immune responses.

To evaluate different routes of immunization, mice were immunizedmucosally (IR, IN, IG) or systemically (SC) with 4 doses (on days 0, 7,14 and 21) of the HIV-1 CLUVAC PCLUS3-18IIIB (SEQ ID NO:2) (50μg/mouse). Thirty-five days after the first dose, antigen-specific Tcells were isolated from PP and SP and tested for the presence of HIV-1P18IIIB—I10 (SEQ ID NO:16) peptide-specific CTL as described above (FIG.7). Closed bars show killing of P18IIIR-I10 (SEQ ID NO:16) pulsedtargets and open bars show killing on unpulsed targets. The level of CTLactivity induced by IR immunization was significantly higher ininductive mucosal tissue (PP) and much higher in systemic immunologicaltissue (SP) than that induced by the other mucosal routes (IN and IG).CTL activity was detected in both PP and SP after immunization via allthree mucosal routes. Systemic immunization (SC) only resulted insignificant CTL activity in SP.

Example 7 CTL Induced by IR Immunization are IFNγ Dependent

Wild-type BALB/c mice and BALB/c mice lacking the ability to producefunctional IFNγ (IFNγ^(−/−) mice) (Dalton et al., Science 259:1739(1993); Tishon et al., Virology 212:244 (1995)) were immunized IR with 4doses (on days 0, 7, 14 and 21) of CLUVAC PCLUS3-18IIIB (SEQ ID NO:2)(50 μg/mouse per immunization) in combination with CT (1 μg/mouse). Onday 35, antigen-specific T cells were isolated from PP, LP and SP.Immune cells from SP, PP, or LP of wild type BALB/c and IFNγ^(−/−) micewere cultured as described above. Cytolytic activity of. CTL wasmeasured (FIG. 8A and FIG. 8B) as described above. The peptidespecificity of CTL was tested as follows: ⁵¹Cr labeled P815 targets werepulsed with peptide P18IIIB-I10 (SEQ ID NO:16) at the beginning of theassay (solid bars), or (as a control) left unpulsed i.e., withoutpeptide (open bars). Induction of both mucosal and systemic CTLresponses by IR immunization was IFNγ dependent, as shown by the lack ofdetectable CTL activity in SP, PP, and LP cells from IFNγ^(−/−) mice(FIG. 8B) and potent CTL activity in SP, PP, and LP cells from wild-typeBALB/c mice (FIG. 8A).

Summarizing the foregoing results, mucosal, administration of anantigenic peptide to mice results in the induction of a protective CTLresponse detectable in both the inductive (Peyer's patch (PP)) andeffector (lamina propria (LP)) sites of the intestinal mucosal immunesystem, as well as in systemic lymphoid tissue, i.e., spleen (SP). Themucosal CTL response is enhanced by co-administering the mucosaladjuvant, cholera toxin (CT) with the antigenic peptide, is inhibited byantibody that specifically binds to (thereby neutralizing the activityof) interleukin-12 (IL-12), and is not detectable in mice lacking theability to produce functional interferon-γ (IFNγ). Furthermore,including IL-12 in the composition of antigenic peptide and CT used forIR immunization resulted in enhanced PP and SP CTL responses relative tothose obtained by IR immunization with antigenic peptide, CT and noIL-12. IR immunization with the viral peptide resulted in protectionfrom viral infection upon subsequent IR challenge with the appropriatevirus.

Example 8 Comparison of Different HIV Vaccine Peptides for use inEliciting Viral Mucosal Protection

Because of the variability of the V3 loop of HIV, further studies wereconducted comparing two cluster peptide constructs using the V3 loop andincorporating the CTL epitope P18 from strain IIIB (Ratner et al.,Nature 313:277-284, (1985)) or MN (Gurgo et al., Virology 164:531-536,(1998)) of HIV-1. These studies were conducted as an exemplary analysisto demonstrate that HIV vaccine cluster peptide constructs can beprepared from different HIV strains and screened in side-by-side assaysto optimize induction of mucosal CTL immunity.

Animals For each of the following Examples, female BALB/c mice werepurchased from Frederick Cancer Research Center (Frederick, Md.).IFNγ^(−/−) mice were purchased from Jackson Laboratories (Bar Harbor,Me.). Mice used in this study were 6-12 weeks old. The IFNγ^(−/−) micewere maintained in a specific pathogen-free microisolator environment.

Immunization Mice were immunized with 4 doses of the synthetic HIVpeptide vaccine construct PCLUS3-18IIIB (Ahlers et al., J. Immunol.150:5647-5665, (1993)) (50 μg/mouse for each immunization) on days 0, 7,14 and 21 in combination with cholera toxin (CT) (10 μg/Imouse) (ListBiological Laboratories, Campbell, Calif.) by intrar tal administration.For subcutaneous imnmunization, incomplete Freund's adjuvant was used.rm IL-12 (a generous gift of Genetics Institute, Inc., Cambridge, Mass.)was delivered either intraperitoneally (IP) (1 μg) or intrarectally (1μg) mixed with DOTAP (Boehringer Mannheim), a cationic lipofectionagent, along with the peptide vaccine.

Cell purification Five weeks to 6 months after the first dose,antigen-specific T cells were isolated from Peyer's patches (PP), laminapropria (LP) and the spleen (SP). The Peyer's patches were carefullyexcised from the intestinal wall and dissociated into single cells byuse of collagenase type VIII, 300 U/ml (Sigma) as described, Mega etal., Int. Immunol. 3:793-805, (1991). Our data showed that most PP CD3⁺T cells isolated from normal mice were CD4⁺, while CD3⁺CD8⁺ T cells wereless frequent. Further, collagenase did not alter expression of CD3,CD4, or CD8 on splenic T cells treated with this enzyme. Lamina proprialymphocyte (LPL) isolation was performed as described, Mega et al., Int.Immunol. 3:793-805, (1991). The small intestines were dissected fromindividual mice and the mesenteric and connective tissues carefullyremoved. Fecal material was flushed from the lumen with un-supplementedmedium (RPMI 1640). After the PP were identified and removed from theintestinal wall, the intestines were opened longitudinally, cut intoshort segments, and washed extensively in RPMI containing 2% fetalbovine serum (FBS). To remove the epithelial cell layer, tissues wereplaced into 100 ml of 1 mM EDTA and incubated twice (first for 40 minand then for 20 min) at 37° C. with stirring. After the EDTA treatment,tissues were washed in complete RPMI medium for 10 min at roomtemperature and then placed into 50 ml of RPMI containing 10% FCS andincubated for 15 min at 370 with stirring. The tissues and medium weretransferred to a 50 ml tube and shaken vigorously for 15 seconds, andthen the medium containing epithelial cells was removed. This mechanicalremoval of cells was repeated twice more, using fresh medium each time,in order to completely remove the epithelial cell layer. Histologicexamination revealed that the structure of the villi and lamina propriawere preserved. To isolate LPL, tissues were cut into small pieces andincubated in RPMI 1640 containing collagenase type VIII 300 U/ml (Sigma)for 50 min at 37° C. with stirring. Supernatants containing cells werecollected, washed and then re-suspended in complete RPMI 1640. Thiscollagenase dissociation procedure was repeated two times and theisolated cells pooled and washed again. Cells were passed through acotton-glass wool column to remove dead cells and tissue debris and thenlayered onto a discontinuous gradient containing 75% and 40% Percoll(Pharmacia Fine Chemicals, Pharmacia Inc., Sweden). After centrifugation(4° C., 600 g, 20 min), the interface layer between the 75% and 40%Percoll was carefully removed and washed with incomplete medium. Thisprocedure provided >90% viable lymphocytes with a cell yield of1.5-2×10⁶ lymphocytes/mouse. The SP were aseptically removed and singlecell suspensions prepared by gently teasing them through sterilescreens. The erythrocytes were lysed in Tris-buffered ammonium chlorideand the remaining cells washed extensively in RPMI 1640 containing 20/oFBS.

Cytotoxic T lvmphocyte assay Immune cells from SP, PP, LP were culturedat 5×10⁶ per/milliliter in 24-well culture plates in complete T cellmedium (CTM): RPMI 1640 containing 10% FBS, 2 mM L-glutamine, penicillin(100 U/ml), streptomycin (100 μg/ml), and 5×10⁻⁵ M 2-mercaptoethanol.Three days later we added 10% concanavalin A supernatant-containingmedium as a source of IL-2. LPL were studied after 7 days stimulationwith IμM P18IIIB-I10 peptide together with 4×10⁶ of 3300-rad irradiatedsyngeneic spleen cells. SP and PP cells were stimulated in vitrosimilarly for one or two 7-day culture periods before assay. Cytolyticactivity of CTL lines was measured by a 4-hour assay with ⁵¹Cr labeledtargets. Two different cell lines were used as a target cells: 1) 15-12cells, Takahashi et al., Proc. Natl. Acad. Sci. USA 85:3105-3109 (1988)(BALB/c 3T3 fibroblasts transfected with HIV-1IIIB gp160 andendogenously expressing HIV gp160), compared with 18 Neo BALB/c 3T3fibroblasts transfected with NeO^(R) alone as a control, and 2) P815targets tested in the presence or absence of I10 peptide (IμM). Fortesting the peptide specificity of ⁵¹Cr labeled P815 targets were pulsedfor 2 hours with peptide at the beginning of the assay. The percentspecific ⁵¹Cr release was calculated as 100× (experimentalrelease-spontaneous release)/(Maximum release-spontaneous release).Maximum release was determined from supernatants of cells that werelysed by addition of 5% Triton-X 100. Spontaneous release was determinedfrom target cells incubated without added effector cells.

Vaccinia virus Recombinant vaccinia virus vPE16 expresses the HIV-1gp160 gene from isolate IIIB (BH8) (Earl et al., J. Virol. 64:2448-2451(1990)). Expression is directed by the compound early/late P7.5promoter. Two copies of the sequence T5NT, which serves as atranscription termination signal for early vaccinia virus genes, arepresent in the IIIB gp 160 gene. Both of these have been altered invPE16, so as to retain the original coding sequence and allow earlytranscription of the gene. The virus, vSC8, is used as a negativecontrol without gp160 (Chakrabarti et al., Mol. Cell Biol. 5:3403-3409(1985)). Both vPE16 and VSC8 express beta-galactosidase.

Determination of virus titer in the ovary On day 35 or 6 months aftercluster peptide HIV vaccine immunization, mice were challengedintrarectally with 2.5×10⁷ or 5×10⁷ pfu of vaccinia virus expressinggp160 IIIB (vPE16). Six days after the challenge with recombinantvaccinia virus expressing HIV-gp160, the mice were killed and ovarieswere removed, homogenized, sonicated, and assayed for vPE16 titer byplating serial 10-fold dilutions on a plate of BSC-2 indicator cellsstaining with crystal violet and counting plaques at each dilution. Theminimal detectable level of virus was 100 pfu.

To compare different HIV vaccine cluster peptide constructs, BALB/c micewere immunized intrarectally with peptide (PCLUS3-18IIIB or PCLUS3-18MN)in the presence of CT as a mucosal adjuvant weekly for four weeks (ondays 0, 7, 14, and 21). Mice were studied either two weeks later (day35) or at six months for memory CTL responses in the Peyer's Patches(PP) or spleen (SP). IR immunization with both peptides PCLUS3-18IIIB orPCLUS3-18MN in combination with CT induces a P18-specific CTL responsein the intestinal PP (FIG. 10, panel A) and in the spleen (FIG. 10,panel B).

However, the level of CTL response after IR immunization withPCLUS3-18MN was significantly lower than after IR immunization withPCLUS3-18IIIB. The difference may reflect a higher affinity of theminimal 10-mer P18IIIB-I10, compared to P18MN-T10, for H-2D^(d)(Takahashi et al., Science 246:118-121 (1989); Takeshita et al., J.Immunol. 154:1973-1986 (1995)). Also, much higher production of IFNγ bymucosal P18IIIB-specific CD8⁺ CTL was observed compared toP18-MN-specific CTL after stimulation with specific peptide in vitro for48 hours. When tested on 15-12 gp160 IIIB-transfected fibroblast targetsendogenously expressing gp 160IIB, the CTL elicited by immunization withPCLUS3-18IIIB also killed these targets. On the basis of theseobservations, the PCLUS3-18IIIB construct was used in the protectionexperiments described below.

Example 9 Mucosal Immunization of Mice with Cluster Peptide ConstructProvides Longlasting Protection from Infection with Recombinant VacciniaVirus Expressing HIVg160

In the foregoing Examples, the ability of the mucosal immune responsesinduced by the HIV cluster peptide vaccine to protect against viruschallenge via a mucosal route is demonstrated. To determine thespecificity of this protection for recombinant protein HIV-1 IIIB gp160,IR immunized mice were challenged on day 35 after the start ofimmunization by IR infusion with vaccinia virus expressing HIV-1 IIIB gp160 (vPE16), or with control vaccinia virus expressing β-galactosidase(vSC8). Unimmunized animals challenged with vPE16 or vSCB served ascontrols. Six days after the challenge, mice were sacrificed and theovaries were removed and assayed for vaccinia titer (6 days afterinfection with vaccinia, the ovaries contain the highest titer ofvirus).

IR immunization with the synthetic HIV peptide vaccine protected miceagainst an IR challenge with vaccinia virus expressing HIV-1 IIIB gp160compared to unimmunized controls, but did not protect against IRchallenge with vaccinia virus expressing only an unrelated protein,β-galactosidase (FIG. 11). Thus, the protection was specific for virusexpressing HIV-1 gp160, and any nonspecific inflammatory responseinduced by the peptide infusion intrarectally was not sufficient toprotect against viral challenge two weeks after the last dose of theimmunization.

Although the presence of mucosal memory CTL precursors was observed,requiring restimulation in vitro for activity 6 months after IRimmunization (Belyakov et al., Proc. Nati. Acad. Sci. 95:1709-1714(1998)), the strength and duration of protection remained unclear. Toresolve this question, the IR immunized mice were challenged 6 monthsafter the start of immunization with PCLUS3-18IIIB by IR administrationwith vaccinia virus expressing HIV-1 IIIB (vPE16). This study showedthat, even 6 months after HIV cluster peptide immunization, BALB/c miceexhibit protection against recombinant HIV-vaccinia challenge (FIG. 12).

Example 10 Protection of Mice Against Mucosal Viral Challenge isMediated by CD8⁺ CTL in the Mucosal Site

To determine the immune mechanism responsible for protection againstmucosal challenge with virus expressing HIV gp160, mice were treated IPwith 0.5 mg monoclonal anti-CD8 antibody (clone 2.43, NIH, Frederick,Md.) one day before and after each of the four immunizations and alsotwo days before and three days after the challenge with vPE16. Thistreatment led to a significant inhibition of the protection againstmucosal challenge with vPE16 (FIG. 13). Thus, protection in the mucosalsite against the virus expressing HIV-1 gp160 is mediated by CD8⁺lymphocytes.

Because the HIV peptide constructs disclosed herein elicit both strongmucosal and systemic MHC class I restricted CD8⁺ CTL responses (Belyakovet al., Proc. Nati. Acad. Sci. 95:1709-1714 (1998)), the role of theseresponses in mediating protection were further investigated. Because SCimmunization with peptide vaccine elicits splenic but not mucosal CTL,whereas IR immunization elicits both (FIG. 14), SC and IR immunizationscan be compared to determine whether systemic CTL are sufficient toprotect against mucosal challenge, or whether local mucosal CTL arenecessary. Accordingly, mice were immunized with PCLUS3-18IIIB plus IFAby the SC route or with PCLUS3-18IIIB and CT by the IR route on days 0,7, 14 and 21, and compared these. On day 35 after the start ofimmunization, these groups of mice as well as unimmunized control micewere challenged by IR administration of vaccinia virus expressing HIV-1gp160 (vPE16). Finally, six days after the challenge, mice weresacrificed and their ovaries assayed for viral titer.

SC immunization with PCLUS3-18IIIB did not protect mice against mucosalchallenge with vPE16, whereas IR immunization with the same peptide didprotect (FIG. 14B). Thus, protection against mucosal challenge withvirus expressing HIV-1 gp160 can be induced only by mucosal immunizationof mice, and correlates with local mucosal CTL activity, not withsplenic CTL activity. On this basis one can conclude that the CD8⁺CTL-mediated protection from mucosal challenge with recombinant vacciniaexpressing HIV-1 gp160 requires local mucosal CD8⁺ CTL, whereas asystemic CTL response is not sufficient.

Example 11 Cytokine Dependence and Enhancement of Protection by LocalAdministration of IL-12 with the Vaccine

Induction of mucosal CTL by peptide vaccine is dependent on endogenousIL-12 in that it can be blocked by in vivo treatment of mice withanti-IL-12 (Belyakov et al., Proc. Nati. Acad. Sci. 95:1709-1714,(1998)). To further define the role of IL-12 in the CTL response andprotection, BALB/c (H-2D^(d)) mice were treated by the IP route with 1μg of the rmIL-12 each day of the IR immunization with PCLUS3-18IIIB (50μg/mice). This treatment did not lead to significant changes in theHIV-specific CTL activity in either mucosal or systemic sites (FIG. 15).However, when the mice were treated with the rmIL-12 (1 μg)+DOTAPintrarectally together with peptide, we found a significant increase inthe CTL level in both mucosal and systemic sites 35 days after the startof immunization (FIG. 15).

In view of the above results, the possibility that rmIL-12 administeredat the local site and time of mucosal immunization might increaseprotection against mucosal challenge with vaccinia virus expressingHIV-1 gp160 was investigated. To address this question, BALB/c mice wereimmunized with the rmIL-12+DOTAP intrarectally together with peptide.The mice were then challenged mice on day 35 after the start ofimmunization by IR administration of vaccinia virus expressing HIV-1IIIBgp160. In this study, twice the dose of challenge virus was used,whereby the unimmunized mice had a titer of several times 10¹⁰ ratherthan several times 10⁸ seen in the previous Examples using a lowerchallenge dose. Nevertheless, the immunized mice showed a reduction ofgreater than 4 logs in virus titer, as had been seen in the earlierexperiments (FIG. 16, bar 2).

Importantly, IR immunization with the synthetic HIV peptide vaccine plusrmIL-12 protected mice against an IR challenge with thisgp-160-recombinant vaccinia virus even more effectively than after theIR immunization with peptide alone (6-log reduction in viral pfu versus4-log reduction, p<0.05) (FIG. 16, bar 3 versus bar 2).

As the induction of mucosal CD8° CTL is strongly dependent on IL-12 andIFNγ (Belyakov et al., Proc. Nati. Acad. Sci. 95:1709-1714 (1998)),further studies were undertaken herein to determine which cytokine actsdirectly if, for the generation of mucosal CTL, and which acts through asecondary mechanism. To address this question, IFNγ^(−/−) mice (BALB/cbackground) and conventional BALB/c mice were treated with the rmIL-12(I μg/mouse)+DOTAP IR together with peptide. Mucosal treatment ofIR-immunized IFNγ^(−/−) mice with rmIL-12 did not lead to the inductionof mucosal or systemic CTL (FIG. 17). It thus appears that IL-12 cannotact directly in the induction of mucosal CD8⁺ CTL in the absence ofIFNγ.

In summary, the foregoing Examples incorporate a novel viral challengesystem in which recombinant vaccinia virus expressing HIV-1 gp160 isused as a surrogate for HIV-1, since we cannot infect the mice withHIV-1. Importantly, in this system, neutralizing antibodies to gp160cannot protect against recombinant vaccinia expressing gp160, becausethe virus does not incorporate gp160 in the virus particle but expressesit only in the infected cell. Thus, the protective immune response mustbe directed at the infected cell.

The results herein demonstrate that the protective response iscompletely dependent on CD8⁺ cells., by the abrogation of protectionafter in vivo depletion of CD8⁺ cells. Thus, the results showunequivocally that it is CD8⁺ CTL (whether via lytic activity or viasecretion of cytokines or other soluble factors) that protect. Since ithas been shown that protection against vaccinia infection can bemediated by interferon-γ which is secreted by CD8⁺ CTL in response toantigen stimulation (Harris et al., J. Virol. 69:910-915 (1995)), it ispossible that the mechanism involves local secretion of this cytokine bythe CTL rather than lysis of infected cell. By either mechanism, the CTLare responsible for mediating protection.

However, since the mucosal immunization induces CTL in both the localmucosal site and the spleen, this result does not distinguish which CTLare responsible for protection. To address this important question, thepresent Examples take advantage of the fact that subcutaneousimmunization with the peptide vaccine induces systemic CTL in the spleenat a level at least as high as that induced by mucosal immunization, butdoes not induce mucosal CTL. Splenic CTL resulting from bothimmunization routes kill target cells endogenously expressing HIV-1gp160. Thus, if systemic CTL against this epitope protected againstmucosal challenge, then the subcutaneously immunized mice would havebeen expected to be protected.

However, the subcutaneously immunized mice showed no evidence ofprotection against mucosal challenge. Thus, the protection correlatedwith CrL activity in the local mucosal sites, Peyer's patches and laminapropria, not with CTL activity in the spleen. The protection was notonly mediated by CTL, but also required CTL in the local mucosal site ofchallenge. Systemic CTL were not sufficient.

Protection mediated by local mucosal CTL appears independentlysufficient to mediate a protective immune response. This conclusion issupported by the observation that a two-log reduction in viral pfuoccurs in the ovary even at day 2 after mucosal viral challenge (from2.37×10⁶ pfu in unimmunized mice to 3.34×10⁴ pfu in IR immunized mice),before much replication could have occurred in the ovary. This findingindicates that the reduction in titer in the ovary reflects a reductionin the amount of virus that can escape the initial mucosal site ofinfection. In addition, the enhancement of CTL activity and protectionby rmIL-12 depended on local mucosal administration of the cytokine, notsystemic administration.

One possible difference between CTL induced by mucosal versus systemicimmunization is that the CTL resulting from the SC immunization do nothave homing receptors for the GI mucosa, as evidenced by the fact thatthey are not detected in the lamina propria or Peyer's patches.

The present disclosure represents the first demonstration of protectionagainst GI mucosal challenge requiring local mucosal CTL at the site ofchallenge. These results have important implications for the developmentof protective vaccines against mucosal exposure to viruses.

In addition to these results, the Examples provided herein alsodemonstrate a surprising persistence, not only of memory CTL in themucosa, but also of protective immunity against mucosal viral challenge.Factors controlling CTL memory, and the role of persistent antigen inmaintaining memory CTL, represent another issue that has been ofinterest for some time (Ahred and Gray, Science 272:54-60 (1996); Kundiget al., Proc. Natl. Acad. Sci. U.S.A. 93:9716-9723 (1996); Ehl et al.,Eur. J. Immunol. 27:3404-3413 (1997)), but has been little studied inthe context of mucosal immune responses and protection. In studies ofsystemic immunity, it was shown (Slifka et al., Blood 90:2103-2108,(1997)) that after infection with lymphocytic choriomeningitis virus(LCMV), CTL memory responses were present in the bone marrow for atleast 325 days, indicating long-term persistence of antiviral T cells atthis site. While the antigen-specific CD8⁺ T cell number droppedprecipitously following viral clearance, substantial numbers persistedfor the life of the mouse (Muraii-Krishna et al., Immunity 8:177-187,(1998)). Upon rechallenge with LCMV, there was rapid expansion of memoryCD8⁺ T cells.

These results indicate that systemic infection with virus can lead tolong-term memory and protection. In the case of mucosal CTL memory, itwas shown that memory CTL remained at the mucosal site longer if theimmunization was via the mucosal route (Gallichan and Rosenthal, J. Exp.Med. 184:1879-1890 (1996)), but the duration of protection by suchmucosal CTL was not studied. The ability of mucosal memory CTL toprotect will depend in part on the rapidity by which they can expand andbe activated after virus exposure, which may relate to the level ofvirus replication in the mucosal site. For these reasons, it isimportant to determine the duration of mucosal protection dependent onlocal mucosal CD8⁺ CTL. The results herein show persistence of CTL evensix months after mucosal immunization with the peptide vaccineconstruct, without additional reimmunization beyond the initialthree-week course. This response is accompanied by protection againstmucosal challenge with vaccinia virus expressing gp160.

This latter result is particularly striking because the exemplaryimmunogen is a peptide administered without any depot form of adjuvantthat would maintain the presence of antigen for extended periods. Itwould be expected that free peptide delivered to the lumen of the gut,or even after transport by mucosal cells, would have a very shorthalf-life. Therefore, either memory CTL can persist in the mucosa atlevels sufficient to mediate protection in the absence of persistentantigen, or antigen must persist locally in some cell-bound form,perhaps on N4HC molecules of dendritic cells. Yet another possibility isthat the peptide crossreacts with one of the antigens in the mucosalflora and that these crossreactive antigens maintain the memory CTL.

Protection against viral infection by CTL involves more than just thenumber of CTL induced. Quality of CTLs is as important for in vivoprotection as quantity. Previous reports have shown that high-avidityP18IIIB-specific CTLs adoptively transferred into severe combinedimmunodeficient (SCID) mice were 100- to 1000-fold more effective atviral clearance than the low-avidity CTLs specific for the samepeptide-MHC complex, despite the fact that all CTL lines lysed virusinfected targets in vitro (Alexander-Miller et al., Proc. Natl. Acad.Sci. U.S.A. 93:4102-4107 (1996)). Thus, the CTL induced by mucosalimmunization with the synthetic peptide vaccine must be present not onlyin sufficient quantity to protect, but also must be of high enoughavidity to protect.

Other aspects of the present disclosure enable optimization of inductionof the CTL response and protective immunity. Delivery of certaincytokines such as IL-12 at the site of antigen immunization systemicallyhave been shown to enhance systemic CTL responses (Ahlers et al., J.Immunol. 159:3947-3958 (1997); Iwasaki et al., J. lmmunol. 159:4591-4601(1997); Xiang and Ertl, Immunity 2:129-135 (1995); Irvine et al., J.Immunol. 156:238-245 (1996)). However, no comparable studies have beenconducted for mucosal CTL responses.

In the model system disclosed herein, induction of mucosal CTL isdependent on endogenous production of IL-12 by the mouse, because itcould be inhibited by anti-IL-12 antibody given in vivo before and aftereach immunization. In the present Examples, IL-12 co-administered withthe antigen intrarectally significantly enhanced CTL induction, and alsoincreased protection against intrarectal vaccinia viral challenge.However, it was striking that IL-12 delivered systemically (i.p.) didnot enhance CTL induction either in the systemic sites (eg. spleen) orin the mucosa. This difference may be due to the short half life ofIL-12 delivered systemically, which prevented it from surviving longenough to get to the sites of CTL induction. Therefore, for mucosal CTLinduction as well as for systemic CTL induction, it is important todeliver the cytokine directly to the site of antigen administration andCTL induction.

Enhancement of CTL induction in the mucosa with recombinant IL-12 is auseful strategy for mucosal vaccine development. In this context, smalldoses given locally in the mucosal sites are not likely to have theglobal toxicity that has been associated with systemic administration ofthis cytokine.

The results herein further show that enhancement of the CTL response invivo by rmIL-12 is dependent on interferon-γ, as no enhancement wasobserved in IFNγ^(−/−) mice. At least two mechanisms can explain thisresult. First, IL-12 may be acting through its well-defined ability toinduce production of IFNγ (Trinchieri, G., Blood 94:4008-4027 (1994)),which then acts directly on CTL precursors. Alternatively, since IFNγ isimportant for expression of the IL-12 receptor (Szabo, et al., J. Exp.Med. 185:817-824 (1997)) IL-12 may act directly on CTL, but may not beable to act in IFNγ^(−/−) mice because of the lack of IL-12R expression.

There is evidence that CTL activity may play a role in protectiveimmunity in humans against HIV-1 (reviewed in (Rowland-Jones et al.,Adv. Immunol. 65:277-346 (1997); Yang, 0.0. and B. D. Walker, Adv.Immunol. 66:273-311 (1997); Berzofsky and Berkower, AIDS 9(A):S 143-S157 (1995)). Cell-mediated immunity to HIV-1 has been demonstrated inuninfected high risk adults (Pinto et al., J. Clin. Invest. 96:867-876,(1995); Rowland-Jones et al., Nature Medicine 1:59-64, (1995)) and inuninfected children born to infected mothers (Clerici et al., AIDS7:1427-1433 (1993); Luzuriaga and Sullivan, J. Cell. Blochem. Supplement17E:98.(Abstract) (1993)). CTL activity has been correlated with lowviral load and long-term non-progressor status in some infectedindividuals (Cao et al., N. Enal. J. Med. 332:201-208 (1995)). CTL havealso been associated with recovery from acute HIV or SIV infection(Yasutomi et al., J. Virol. 67:1707-1711 (1993); Reimann et al., J.Virol. 68:2362-2370 (1994); Koup et al. J. Virol. 68:4650-4655 (1994);Borrow et al., Nature Medicine 3:205-211, (1997)). Induction of escapemutations by CTL implies that the CTL are eliminating the bulk of thewild type virus (Borrow et al., Nature Medicine 3:205-211 (1997);Phillips et al., Nature 354:453-459 (1991); Nowak et al., Nature375:606-611 (1995); Goulder et al., Nature Medicine 3:212-217 (1997);Couillin et al., J. Exp. Med. 180:1129-1134 (1994); Koenig et al.,Nature Medicine 1:330-336 (1995)). In addition, in an SIV study,protection was associated with a particular simian class I MHC molecule(Heeney et al., J. Exp. Med. 180:769-774, (1994)). CD8⁺ cells have beendemonstrated to suppress replication of human and simian lentiviruses inautologous CD4 cells by a non-lytic mechanism involving soluble factorssynthesized and released by activated CD8⁺ cells (Walker et al., Science234:1563-1566 (1986); Tsubota et al., J. Exp. Med. 169:1421-1434 (1989);Walker and Levy, Immunology 66:628-630 (1989); Mackewicz et al., J.Clin. Invest. 87:1462-1466, (1991); van Kuyk et al., J. Immunol.153:4826-4833 (1994)). Such soluble factors include the chemokinesRANTES, MIP-1α, and MIP-1β (Cocchi et al., Science 270:1811-1815(1995)).

The present disclosure provides the first demonstration of protectionagainst mucosal viral challenge mediated by CTL, which must be presentin the local mucosa. Also provided are methods and compositions forenhancing the; induction of such local mucosal CTL by a peptide vaccinegiven together with recombinant IL-12 at the mucosal site. These methodsand compositions for stimulating CTL immunity at local sites of mucosalexposure represent important tools for vaccine development to preventHIV infection or disease in humans. Because the gastrointestinal tractappears to be a major site of early SIV and HIV replication, among otherpathogens, and because this and other mucosal sites are frequent sitesof entry for these pathogens, it is particularly critical to achieveprotection at mucosal sites. The present disclosure satisfies theseobjects and provides other advantages as set forth above.

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

20 1 35 PRT Human immunodeficiency virus type 1 1 Glu Gln Met His GluAsp Ile Ile Ser Leu Trp Asp Gln Ser Leu Lys 1 5 10 15 Pro Cys Val LysArg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Thr 20 25 30 Ile Gly Lys 352 39 PRT Human immunodeficiency virus type 1 2 Lys Gln Ile Ile Asn MetTrp Gln Glu Val Gly Lys Ala Met Tyr Ala 1 5 10 15 Pro Pro Ile Ser GlyGln Ile Arg Arg Ile Gln Arg Gly Pro Gly Arg 20 25 30 Ala Phe Val Thr IleGly Lys 35 3 39 PRT Human immunodeficiency virus type 1 3 Arg Asp AsnTrp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile 1 5 10 15 Glu ProLeu Gly Val Ala Pro Thr Arg Ile Gln Arg Gly Pro Gly Arg 20 25 30 Ala PheVal Thr Ile Gly Lys 35 4 48 PRT Human immunodeficiency virus type 1 4Ala Val Ala Glu Gly Thr Asp Arg Val Ile Glu Val Val Gln Gly Ala 1 5 1015 Tyr Arg Ala Ile Arg His Ile Pro Arg Arg Ile Arg Gln Gly Leu Glu 20 2530 Arg Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Thr Ile Gly Lys 35 4045 5 42 PRT Human immunodeficiency virus type 1 5 Asp Arg Val Ile GluVal Val Gln Gly Ala Tyr Arg Ala Ile Arg His 1 5 10 15 Ile Pro Arg ArgIle Arg Gln Gly Leu Glu Arg Arg Ile Gln Arg Gly 20 25 30 Pro Gly Arg AlaPhe Val Thr Ile Gly Lys 35 40 6 30 PRT Human immunodeficiency virus type1 6 Asp Arg Val Ile Glu Val Val Gln Gly Ala Tyr Arg Ala Ile Arg Arg 1 510 15 Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Thr Ile Gly Lys 20 25 30 732 PRT Human immunodeficiency virus type 1 7 Ala Gln Gly Ala Tyr Arg AlaIle Arg His Ile Pro Arg Arg Ile Arg 1 5 10 15 Arg Ile Gln Arg Gly ProGly Pro Arg Ala Phe Val Thr Ile Gly Lys 20 25 30 8 35 PRT Humanimmunodeficiency virus type 1 8 Glu Gln Met His Glu Asp Ile Ile Ser LeuTrp Asp Gln Ser Leu Lys 1 5 10 15 Pro Cys Val Lys Arg Ile His Ile GlyPro Gly Arg Ala Phe Tyr Thr 20 25 30 Thr Lys Asn 35 9 39 PRT Humanimmunodeficiency virus type 1 9 Lys Gln Ile Ile Asn Met Trp Gln Glu ValGly Lys Ala Met Tyr Ala 1 5 10 15 Pro Pro Ile Ser Gly Gln Ile Arg ArgIle His Ile Gly Pro Gly Arg 20 25 30 Ala Phe Tyr Thr Thr Lys Asn 35 1039 PRT Human immunodeficiency virus type 1 10 Arg Asp Asn Trp Arg SerGlu Leu Tyr Lys Tyr Lys Val Val Lys Ile 1 5 10 15 Glu Pro Leu Gly ValAla Pro Thr Arg Ile His Ile Gly Pro Gly Arg 20 25 30 Ala Phe Tyr Thr ThrLys Asn 35 11 48 PRT Human immunodeficiency virus type 1 11 Ala Val AlaGlu Gly Thr Asp Arg Val Ile Glu Val Val Gln Gly Ala 1 5 10 15 Tyr ArgAla Ile Arg His Ile Pro Arg Arg Ile Arg Gln Gly Leu Glu 20 25 30 Arg ArgIle His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr Lys Asn 35 40 45 12 42PRT Human immunodeficiency virus type 1 12 Asp Arg Val Ile Glu Val ValGln Gly Ala Tyr Arg Ala Ile Arg His 1 5 10 15 Ile Pro Arg Arg Ile ArgGln Gly Leu Glu Arg Arg Ile His Ile Gly 20 25 30 Pro Gly Arg Ala Phe TyrThr Thr Lys Asn 35 40 13 30 PRT Human immunodeficiency virus type 1 13Asp Arg Val Ile Glu Val Val Gln Gly Ala Tyr Arg Ala Ile Arg Arg 1 5 1015 Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr Lys Asn 20 25 30 1431 PRT Human immunodeficiency virus type 1 14 Ala Gln Gly Ala Tyr ArgAla Ile Arg His Ile Pro Arg Arg Ile Arg 1 5 10 15 Arg Ile His Ile GlyPro Gly Arg Ala Phe Tyr Thr Thr Lys Asn 20 25 30 15 15 PRT Humanimmunodeficiency virus type 1 15 Arg Ile Gln Arg Gly Pro Gly Arg Ala PheVal Thr Ile Gly Lys 1 5 10 15 16 10 PRT Human immunodeficiency virustype 1 16 Arg Gly Pro Gly Arg Ala Phe Val Thr Ile 1 5 10 17 20 PRT Humanimmunodeficiency virus type 1 17 Glu Gln Met His Glu Asp Ile Ile Ser LeuTrp Asp Gln Ser Leu Lys 1 5 10 15 Pro Cys Val Lys 20 18 24 PRT Humanimmunodeficiency virus type 1 18 Lys Gln Ile Ile Asn Met Trp Gln Glu ValGly Lys Ala Met Tyr Ala 1 5 10 15 Pro Pro Ile Ser Gly Gln Ile Arg 20 1924 PRT Human immunodeficiency virus type 1 19 Arg Asp Asn Trp Arg SerGlu Leu Tyr Lys Tyr Lys Val Val Lys Ile 1 5 10 15 Glu Pro Leu Gly ValAla Pro Thr 20 20 33 PRT Human immunodeficiency virus type 1 20 Ala ValAla Glu Gly Thr Asp Arg Val Ile Glu Val Val Gln Gly Ala 1 5 10 15 TyrArg Ala Ile Arg His Ile Pro Arg Arg Ile Arg Gln Gly Leu Glu 20 25 30 Arg

What is claimed is:
 1. A method for inducing an antigen specificsystemic and rectal mucosal cytotoxic T lymphocyte (CTL) response in amammalian subject comprising contacting a rectal mucosal tissue of thesubject with a composition comprising a chimeric peptide having theamino acid sequence KQIINMWQEVGKAMYAPPISGQIRIQRGPGRAFVTIGK (SEQ ID NO:2).
 2. The method of claim 1, wherein said composition further comprisesan adjuvant.
 3. The method of claim 2, wherein the adjuvant is selectedfrom cholera toxin (CT), mutant cholera toxin (MCT), or mutant-E. coliheat labile enterotoxin (MLT).
 4. The method of claim 1, furthercomprising administering a purified cytokine to the subject.
 5. Themethod of claim 4, wherein the cytokine is contacted with the rectalmucosal surface.
 6. The method of claim 4, wherein the purified cytokineis selected from granulocyte-macrophage colony-stimulating factor(GM-CSF), interleukin-2 (IL-2), interlukin-7 (IL-7), interleukin-12(IL-12) or tumor necrosis factor a (TNFa).
 7. The method of claim 1,further comprising administering purified interferon-γ to the subject.8. The method of claim 7, wherein the purified interferon-γ is contactedwith the rectal mucosal surface of the subject.
 9. The method of claim4, further comprising administering purified interferon-γ to thesubject.
 10. The method of claim 9, wherein the purified interferon-γ iscontacted with the rectal mucosal surface of the subject.
 11. The methodof claim 1, wherein said composition further comprises a purifiedcytokine selected from granulocyte-macrophage colony-stimulating factor(GM-CSF), interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12(IL-12) or tumor necrosis factor.
 12. The method of claim 1, whereinsaid composition further comprises purified interferon-γ.
 13. The methodof claim 11, wherein said composition further comprises purifiedinterferon-γ.
 14. A method for inducing an antigen specific systemic andrectal mucosal CTL response in a mammalian subject, comprisingcontacting a rectal mucosal tissue of the subject with a compositioncomprising a chimeric peptide having the amino acid sequenceKQIINMWQEVGKAMYAPPISGQIRRIQRGPGR AFVTIGK (SEQ ID NO: 2), wherein saidcomposition does not comprise an adjuvant.
 15. The method of claim 14,further comprising administering a purified cytokine the subject. 16.The method of claim 15, wherein the cytokine is contacted with therectal mucosal surface of the subject.
 17. The method of claim 16,wherein the purified cytokine is selected from granulocyte-macrophagecolony-stimulating factor (GM-CSF, interleukin-2 (IL-2), interleukin-7(IL-7), interleukin-12 (IL-12) or tumor necrosis factor a (TNFa). 18.The method of claim 14, further comprising administering purifiedinterferon-γ to the subject.
 19. The method of claim 18, wherein thepurified interferon-γ is contacted with a mucosal surface of thesubject.
 20. The method of claim 15, further comprising administeringpurified interferon-γ to the subject.
 21. The method of claim 20,wherein the purified interferon-γ is contacted with a mucosal surface ofthe subject.
 22. The method of claim 14, wherein said compositionfurther comprises a purified cytokine selected fromgranulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2(IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12) or tumor necrosisfactor.
 23. The method of claim 14, wherein said composition furthercomprises purified interferon-γ.
 24. The method of claim 22, whereinsaid composition further comprises purified interferon-γ.
 25. Animmunogenic composition comprising a chimeric peptide having the aminoacid sequence KQITNMWQEVGKAMYAPPISGQIRRIQRGPGRAFVTIGK (SEQ ID NO: 2),formulated for intrarectal delivery to the rectum, colon, sigmoid colon,or distal colon; wherein said composition is formulated as a rectalemulsion, foam, suppository, or gel preparation and comprises a base,carrier, or absorption-promoting agent adapted for intrarectal delivery.26. The immunogenic composition of claim 25, wherein the chimericpeptide is admixed with a rectally-compatible homogeneous gel carrier.27. The immunogenic composition of claim 26, wherein the homogenous gelcarrier is a polyoxyethylene gel.
 28. The immunogenic composition ofclaim 25, wherein the suppository is comprised of a base selected from apolyethyleneglycol, witepsol H15, witepsol W35, witepsol E85,propyleneglycol dicaprylate (Sefsol 228), Miglyol 810,hydroxypropylcellulose-H (HPC), or carbopol-934P (CP).
 29. Theimmunogenic composition of claim 28, comprising at least two basematerials.
 30. The immunogenic composition of claim 25, furthercomprising a stabilizing agent to minimize intrarectal degradation ofthe chimeric peptide.
 31. The immunogenic composition of claim 25,wherein the absorption-promoting agent is selected from a surfactant,mixed micelle, enamines, nitric oxide donor, sodium salicylate, glycerolester of acetoacetic acid, clyclodextrin or beta-cyclodextrinderivative, or medium-chain fatty acid.
 32. The immunogenic compositionof claim 25, further comprising an adjuvant.
 33. The immunogeniccomposition of claim 32, wherein the adjuvant is selected from choleratoxin (CT), mutant cholera toxin (MCT), mutant-E. coli heat labileenterotoxin, or pertussis toxin.
 34. The immunogenic composition ofclaim 32, wherein the adjuvant is conjugated to a mucosal tissue or Tcell binding agent.
 35. The immunogenic composition of claim 34, whereinthe mucosal tissue or T cell binding agent is selected from protein A,an antibody that binds a mucosal tissue- or T-cell-specific protein, ora ligand or peptide that binds a mucosal tissue- or T-cell-specificprotein.
 36. The immunogenic composition of claim 32, wherein theadjuvant comprises a recombinant cholera toxin (CT) having a B chain ofCT substituted by protein A conjugated to a CT A chain.
 37. Theimmunogenic composition of claim 32, wherein the adjuvant is conjugatedto a protein or peptide that binds specifically to T cells.
 38. Theimmunogenic composition of claim 32, further comprising purified IL-12.39. The immunogenic composition of claim 32, further comprising purifiedinterferon-γ.
 40. The immunogenic composition of claim 39, furthercomprising purified IL-12.